Methods of cardiac repair

ABSTRACT

Provided herein is a new method to isolate and expand cardiac progenitor/stem cells from a placenta, which produces a cell population enriched in multipotent functional progenitor/stem cells. Cardiac progenitor/stem cells isolated by this method maintain their self-renewal character in vitro and differentiate into normal cells in myocardium, including cardiomyocytes, endothelial cells, and smooth muscle cells, after transplantation into ischemic hearts. Also provided in this application are substantially pure populations of multipotent cardiac progenitor/stem cells, and their use to treat and prevent diseases and injuries, including those resulting from myocardial infarction. A model for assessing the potential of cardiac stem cells for treatment of myocardial infarction is also provided.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/556,700, filed Nov. 7, 2011, which application is incorporated hereinby reference in its entirety.

STATEMENT OF FEDERAL FUNDING

Embodiments of the present application were made, in part, with U.S.government support under NHLBI (R01-HL 088255) awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Heart failure is the leading cause of hospitalization in the US andheart disease remains the number one killer in the industrialized world.Each year over 1.1 million Americans have a myocardial infarction(“MI”), typically caused by a heart attack, with median survival afteronset only 1.7 years in men and 3.2 years in women. Typically, 225,000people suffering MI die before reaching the hospital.

Myocardial infarctions result in an immediate depression in ventricularfunction and the infarctions may expand, thereby causing ventricularremodeling. In many patients, progressive myocardial infarct expansionand ventricular remodeling leads to deterioration of ventricularfunction and heart failure.

A myocardial infarction (MI) occurs when a coronary artery becomesoccluded and can no longer supply blood to the myocardial tissue. When aMI occurs, the myocardial tissue that is no longer receiving adequateblood flow dies and is replaced with scar tissue. Within seconds of amyocardial infarction, the under-perfused myocardial cells no longercontract, leading to abnormal wall motion, high wall stresses within andsurrounding the infarct, and depressed ventricular function. The infarctexpansion and ventricular remodeling are caused by these high stressesat the junction between the infarcted tissue and the normal myocardium.These high stresses eventually kill or severely depress function in thestill viable myocardial cells. Thus, a wave of dysfunctional tissueexpands from the original myocardial infarct region.

The consequences of MI may be often severe and disabling. In addition toimmediate hemodynamic effects, the infarcted tissue and the myocardiumor cardiac tissue undergo three major processes: infarct expansion,infarct extension, and ventricular remodeling. The magnitude of theresponses and the clinical significance relates to the size and locationof the myocardial infarction (Weisman and Healy, “Myocardial InfarctExpansion, Infarct Extension, and Reinfarction: PathophysiologicalConcepts”, Progress in Cardiovascular Disease 1987; 30:73-110; Kelley etal., “Restraining Infarct Expansion Preserves Left Ventricular Geometryand Function After Acute Anteroapical Infarction,” Circulation 1999, 99:135-142). Myocardial infarctions that destroy a higher percentage of thenormal myocardium, and myocardial infarctions that are locatedanteriorly on the heart generally become clinically significant.

SUMMARY OF THE INVENTION

Disclosed herein is a new approach towards the regeneration and repairof cardiac myocytes. The disclosed compositions and methods can be usedin various clinical applications.

Provided herein is a composition comprising a population of cells and apharmaceutically acceptable carrier for increasing cardiomyocyteformation, increase cardiomyocyte proliferation, increase cardiomyocytecell cycle activation, increase mitotic index of cardiomyocytes,increase myofilament density, increase borderzone wall thickness, or acombination thereof, wherein said cells express one or more markersidentified in Table 2 or in FIG. 5C. In one embodiment, the cells arederived from placenta. In another embodiment, the cells are progenitorcells or stem cells. In another embodiment, the cells express Cdx2, Cd9,Eomes, CD34, CD31, c-kit, or a combination thereof. In anotherembodiment, the cells express Cdx2 and Cd9.

Also provided herein is a composition comprising a population of cellsand a pharmaceutically acceptable carrier for treating myocardialinfarction, chronic coronary ischemia, arteriosclerosis, congestiveheart failure, dilated cardiomyopathy, restenosis, coronary arterydisease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, or myocardial hypertrophy, wherein said cells express oneor more markers identified in Table 2 or in FIG. 5C. In one embodiment,the cells are derived from placenta. In another embodiment, the cellsare progenitor cells or stem cells. In another embodiment, the cellsexpress Cdx2, Cd9, Eomes, CD34, CD31, c-kit, or a combination thereof.In another embodiment, the cells express Cdx2 and Cd9.

Also provided herein is a composition comprising a population of cellsand a pharmaceutically acceptable carrier for inducing cardiacregeneration, wherein said cells express one or more markers identifiedin Table 2 or in FIG. 5C. In one embodiment, the cells are derived fromplacenta. In another embodiment, the cells are progenitor cells or stemcells. In another embodiment, the cells express Cdx2, Cd9, Eomes, CD34,CD31, c-kit, or a combination thereof. In another embodiment, the cellsexpress Cdx2 and Cd9. In another embodiment, the composition increasescardiomyocyte formation, increase cardiomyocyte proliferation, increasecardiomyocyte cell cycle activation, increase mitotic index ofcardiomyocytes, increase myofilament density, increase borderzone wallthickness, or a combination thereof, when administered to a subject. Inanother embodiment, the composition treats myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy when administered to a subject.

One aspect of the invention is directed to a method for restoringcardiac function. In such methods an effective amount of a compositionthat includes Cdx2 stem cells and/or Cdx2 progenitor cells is introducedinto the heart of a subject in need thereof. The Cdx2 cells can be anisolated Cdx2 cell population. In one embodiment, such cells areisolated from placental tissue. In addition to Cdx2 cells, thecomposition may also include various pharmaceutically acceptablecarriers and/or adjuvants as described herein.

Another aspect provided herein is a method for restoring cardiacfunction comprising introducing an effective amount of a compositioncells and a pharmaceutically acceptable carrier into a heart of asubject in need thereof, wherein said cells express one or more markersidentified in Table 2 or in FIG. 5C. Also provided herein is a method ofinducing cardiomyocyte regeneration, cardiac repair, vasculogenesis orcardiomyocyte differentiation, comprising contacting cells with injuredheart tissue, wherein said cells express one or more markers identifiedin Table 2 or in FIG. 5C.

In one embodiment, the cells are derived from placenta. In anotherembodiment, the cells are progenitor cells or stem cells. In anotherembodiment, the cells express Cdx2, Cd9, Eomes, CD34, CD31, c-kit, or acombination thereof. In another embodiment, the cells express Cdx2 andCd9.

Generally, a subject upon which the methods of the invention are to beperformed will have been diagnosed with myocardial infarction, chroniccoronary ischemia, arteriosclerosis, congestive heart failure, dilatedcardiomyopathy, restenosis, coronary artery disease, heart failure,arrhythmia, angina, atherosclerosis, hypertension, or myocardialhypertrophy. Alternatively, it will have been determined that a subjectupon which the methods of the invention are performed is at risk formyocardial infarction, chronic coronary ischemia, arteriosclerosis,congestive heart failure, dilated cardiomyopathy, restenosis, coronaryartery disease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, or myocardial hypertrophy based on assessment of the hearttissue and/or family history. In one embodiment, a subject has beendiagnosed with myocardial infarction or at risk for heart failure.

Compositions described herein may be implanted into cardiac tissue ofthe subject. Alternatively, implantation can be via injection deliveryor catheter-delivery.

The cardiac tissue into which the composition is introduced can bemyocardium, endocardium, epicardium, connective tissue in the heart, ornervous tissue in the heart.

In various embodiments, the subject is an animal (e.g., a mammal) suchas, for example, a human, a rodent (e.g., mice, rats, etc.), a primate(e.g., a gorilla, a chimpanzee, an orangutan, a monkey, etc.), aveterinary animal (e.g., a horse, a bull, a cow, a sheep, a pig, etc.),a domestic animal (e.g., a dog, a cat, etc.), a reptile, avians (e.g.,chickens or turkeys, etc.), or any other animal in need of suchtreatment.

In various embodiments, the cell population increases cardiomyocyteformation, increases cardiomyocyte proliferation, increasescardiomyocyte cell cycle activation, increases mitotic index ofcardiomyocytes, increases myofilament density, increases borderzone wallthickness, or a combination thereof.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for the preparation of amedicament for inducing cardiac regeneration. In one embodiment, thecells are derived from placenta. In another embodiment, the cells areprogenitor cells or stem cells. In another embodiment, the cells expressCdx2, and further express Cd9, Eomes, CD34, CD31, c-kit, or acombination thereof. In another embodiment, the cells express Cdx2 andCd9.

In one embodiment, the composition increases cardiomyocyte formation,increase cardiomyocyte proliferation, increase cardiomyocyte cell cycleactivation, increase mitotic index of cardiomyocytes, increasemyofilament density, increase borderzone wall thickness, or acombination thereof, when administered to a subject.

In another embodiment, the composition treats myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy when administered to a subject.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for the preparation of amedicament to increase cardiomyocyte formation, increase cardiomyocyteproliferation, increase cardiomyocyte cell cycle activation, increasemitotic index of cardiomyocytes, increase myofilament density, increaseborderzone wall thickness, or a combination thereof. In one embodiment,the cells are derived from placenta. In another embodiment, the cellsare progenitor cells or stem cells. In another embodiment, the cellsexpress Cdx2, and further express Cd9, Eomes, CD34, CD31, c-kit or acombination thereof. In another embodiment, the cells express Cdx2 andCd9.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for the preparation of amedicament for treating myocardial infarction, chronic coronaryischemia, arteriosclerosis, congestive heart failure, dilatedcardiomyopathy, restenosis, coronary artery disease, heart failure,arrhythmia, angina, atherosclerosis, hypertension, or myocardialhypertrophy.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for inducing cardiacregeneration. In one embodiment, the cells are derived from placenta. Inanother embodiment, the cells are progenitor cells or stem cells. Inanother embodiment, the cells express Cdx2, and further express Cd9,Eomes, CD34, CD31, c-kit, or a combination thereof. In anotherembodiment, the cells express Cdx2 and Cd9.

In one embodiment, the composition increases cardiomyocyte formation,increases cardiomyocyte proliferation, increases cardiomyocyte cellcycle activation, increases mitotic index of cardiomyocytes, increasesmyofilament density, increases borderzone wall thickness, or acombination thereof, when administered to a subject.

In another embodiment, the composition treats myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy when administered to a subject.

A composition including a population of Cdx2 cells and apharmaceutically acceptable carrier to increase cardiomyocyte formation,increase cardiomyocyte proliferation, increase cardiomyocyte cell cycleactivation, increase mitotic index of cardiomyocytes, increasemyofilament density, increase borderzone wall thickness, or acombination thereof. In one embodiment, the cells are derived fromplacenta. In another embodiment, the cells are progenitor cells or stemcells. In another embodiment, the cells express Cdx2, and furtherexpress Cd9, Eomes, CD34, CD31, c-kit, or a combination thereof. Inanother embodiment, the cells express Cdx2 and Cd9.

A composition including a population of Cdx2 cells and apharmaceutically acceptable carrier for treating myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy. In one embodiment, the cells are derived fromplacenta. In another embodiment, the cells are progenitor cells or stemcells. In another embodiment, the cells express Cdx2, and furtherexpress Cd9, Eomes, CD34, CD31, c-kit, or a combination thereof. Inanother embodiment, the cells express Cdx2 and Cd9.

One would understand that one or more additional excipients, carriers,adjuvants, cells, etc., may be added to the compositions describedherein.

Compositions described herein may contain, for example, from about 1×10⁸to about 1×10² cells. Compositions may also contain, for example, fromabout 1×10⁶ to about 1×10⁵ cells. In one embodiment where additionalcells are present in the composition, the amount of cells in thecomposition will generally contain about 1×10⁸ to about 1×10² Cdx2cells. In another embodiment, where additional cells are present in thecomposition, the composition will generally contain about 1×10⁶ to about1×10⁵ Cdx2 cells.

Provided herein is a mouse model to study myocardial infarction and therole of fetal cells in treatment of cardiac injury. The model comprises(1) mating wild-type female mice with eGFP positive male mice to formeGFP positive fetuses; (2) inducing myocardial infarction in thepregnant mice at E12 days; (3) assessing maternal hearts for eGFPpositive cells, wherein the presence of eGFP positive cells indicatesmigration of fetal cells to the material heart and/or assessing one ormore symptoms of myocardial infarction. In one embodiment eGFP positivecells have differentiated and have formed endothelial cells, smoothmuscle cells and/or cardiomyocytes.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the compositions and methods are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of the present embodiments will be obtained byreference to the following detailed description that sets forthillustrative examples, in which the principles of the compositions andmethods are utilized, and the accompanying drawings of which:

FIGS. 1A-D. Experimental model and tracking of eGFP+ fetal cells inmaternal heart. FIG. 1A) Schematic of the experimental protocol. FIG.1B) Mice were sacrificed at several time points for molecular andcellular analyses to track eGFP+ cells in maternal hearts and assesstheir differentiation pathways. FIGS. 1C-D) Quantitative PCRdemonstrates significantly greater levels of eGFP expression in pregnantmice subjected to cardiac injury {(1 week: 120.0±17.0; FIG. 1C) (2weeks: 12.0±1.6; FIG. 1D), n=3} compared to shams {(1 week: 6.0±1.7) (2weeks: 1.6±0.4), n=3} and non-infarcted controls {(1 week: 1.0±0.6) (2weeks: 1.0±0.7), n=3}, error bars are standard error of the mean(s.e.m.).

FIG. 2. Fetal cells differentiate into diverse cardiac lineages afterhoming to maternal heart. Mean intensities of the spectral profiles fromROIs 1-6 where ROIs 1, 2, and 6 are control areas and ROIs 3, 4, and 5represent eGFP+ cells.

FIGS. 3A-B. Fetal cells exhibit clonality and undergo cardiacdifferentiation in a fusion-independent manner. FIG. 3A) Single cellsorting of eGFP+ fetal cells from maternal hearts into 96-well platesdemonstrates clonal expansion with a clonal efficiency of −8.3% onfeeder cell layers made with WT neonatal cardiomyocytes. FIG. 3B) Thenumber of cells on each day after initial plating is provided for eachsample.

FIGS. 4A-C. Fetal cells selectively home to injured maternal hearts andnot to non-injured organs; fetal cells express various stem cellmarkers, including Cdx2. FIG. 4A) eGFP+ cells were sorted from cellsuspensions prepared from various organs and tissues. FIG. 4B) Fetalcell numbers in injured heart and blood increased immediately afterdelivery. Representative FACS profiles are shown for eGFP+ cell sortingfrom injured heart, blood and non-injured organs with mean percentagesof eGFP+ cells (minimum n=3). FIG. 4C) Mean percentages of fetal cellsplus s.e.m. plotted for each organ as follows: MI Heart-1.10±0.90 beforedelivery (n=10), 6.32±0.90 after delivery (n=19), p=0.001;Blood-1.34±0.81 before delivery (n=10), 3.59±2.30 after delivery (n=15),p=NS; Placenta-35.6±11.47 after delivery (n=3). Very low to undetectablenumbers are found for all other organs.

FIGS. 5A-C. Fetal cells selectively homing to injured maternal heartsexpress various stem cell markers, including Cdx2. FIG. 5A) eGFP+ fetalcells were sorted from maternal hearts one week after injury.Percentages of eGFP+ cells expressing various stem/progenitor cellsurface markers and transcription factors were quantitated using FACSanalysis. FIG. 5B) Quantitation for each marker above was performed intriplicate and mean percentage plus s.e.m. was plotted as follows:Nkx2.5 79.69±8.08; CD31 46.40±8.77; Cdx2 38.39±5.70; Sca-1 20.73±0.80;c-Kit 25.39±2.99; Islet1 2.99±0.97; Pou5f1 1.97±0.82; Nanog 2.72±0.49;Sox2 23.52±1.85; CD34 14.90±2.97. FIG. 5C) eGFP+ cells were sorted fromend-gestation placentas from three pregnant mice subjected to myocardialinjury. RNA expression array of 92 pluripotency genes was performed;gene expression relative to GAPDH was plotted for genes with the highestexpression levels. Of note, TS cell markers Cdx2 and Eomes are amongstgenes with highest expression.

FIG. 6. Model depicting trafficking of cells from fetus across placentainto maternal circulation to injury and peri-injury zones of thematernal heart. Cells of fetal origin engraft within maternal heart andgive rise to diverse cardiac lineages including cardiomyocytes, smoothmuscle cells and endothelial cells.

FIG. 7. Negligible Nkx2.5 expression was observed in late term placentaof mouse with cardiac injury relative to positive control (E16.5 heart).Nkx2.5 expression by q-PCR above was plotted relative to Nkx2.5expression in E16.5 heart.

FIG. 8. Absolute quantification of GFP cells in whole hearts of pregnantfemale mice mated with GFP-transgenic males is shown. Standard curveswere generated for both GFP and ApoB by plotting CT values for differentquantities of known amounts of DNA from GFP transgenic mice versus theDNA quantity in nanograms (ng). In the first row, data for the 2 weekstime point are presented; in the second row, data for the 1 week timepoint are presented. Column A represents time point; Column B representssample type; Column C depicts CT value (in triplicate averaged over 3mice) for each sample; Column D represents the DNA quantity asextrapolated from the GFP standard curve for each experimental CT value;Column E is the DNA quantity as extrapolated from the ApoB standardcurve; Column F is the ratio of values in E/values in D (normalizing toApoB expression levels as described in learn.appliedbiosystems.com);Column G represents the inverse log of values in F to derive‘normalized’ DNA quantity; Column H is the DNA quantity converted to pg;Column I is the number of GFP cells in that sample of DNA utilizing themouse genome conversion factor for this strain of mouse as referred toin Fujiki et al., Biol. Reprod, 2008 and Column J represents theabsolute percentage of GFP cells in the whole heart-1.3% cells of wholeheart are GFP-positive at 1 week post-injury and 1.7% cells of wholeheart are GFP-positive at 2 weeks post-injury.

FIG. 9. The results of Real time q-PCR. Nkx2.5 gene expression in lateterm placenta of mouse with cardiac injury are shown relative to thepositive control (E16.5 mouse heart).

FIG. 10. FIG. 10A illustrates a lentivirus with murine Cdx2 promoterdriving expression of tdTomato that will be used for selecting Cdx2cells from placenta tissues. FIG. 10B illustrates a control lentivirushaving the same backbone as in 10 A, but utilizing a CMV promoter todrive expression of tdTomato. FIG. 10C illustrates flow cytometricanalysis of lentivirual transduction in CT26. Wild type (WT) murinecolon carcinoma cell line.

DETAILED DESCRIPTION OF THE INVENTION

The studies described herein were inspired by the clinical observationthat women with peripartum cardiomyopathy exhibit the highest rate ofrecovery amongst all known etiologies of heart failure. The presentinventors postulated that fetal cells may contribute to this recoveryand, therefore, created a new mouse model of experimental cardiac injuryin pregnant females carrying eGFP-tagged fetuses.

Fetal cells enter the maternal circulation during pregnancy and maypersist in maternal tissue for decades as microchimeras. Fetal maternaltransfer of cells can involve multiple cell types, some withregenerative properties, but this phenomenon had not been previouslyexplored in acute cardiac injury.

The present inventors determined for the first time that fetal cellsselectively home (migrate) to injured maternal hearts and undergodifferentiation into diverse cardiac lineages. Utilizing enhanced greenfluorescent protein (eGFP)-tagged fetuses, engraftment of multipotentfetal cells in injury zones of maternal hearts was demonstrated. Invivo, eGFP+ fetal cells were found to form endothelial cells, smoothmuscle cells, and cardiomyocytes. In vitro, fetal cells isolated frommaternal hearts recapitulate these differentiation pathways,additionally forming vascular tubes and beating cardiomyocytes in afusion-independent manner. About 40% of fetal cells in the maternalheart were found to express Caudal-related homeobox2 (Cdx2).

Fetal maternal stem cell transfer was found to have an effect onmaternal response to cardiac injury. Furthermore, Cdx2 cells wereidentified as a novel cell type for cardiovascular regenerative therapy.

The presently disclosed findings demonstrate for the first time thatfetal cells selectively home to injured heart tissue and undergodifferentiation into diverse cardiac lineages in vivo and in vitro. Whenfetal cells are isolated from the maternal heart, they form beatingcardiomyocytes in vitro, in addition to forming vascular tubes, smoothmuscle cells, and endothelial cells. Approximately 40% of the fetalcells entering the maternal heart are Cdx2-positive. Cdx2 has previouslybeen known as a marker of trophoblast stem (TS) cells that give rise toplacenta but not other organs.

The results of the studies described herein provide several newdiscoveries. Firstly, the phenomenon of fetal-maternal stem celltransfer has never previously been explored in the realm of acutecardiovascular disease. The present inventors determined that the fetalcells “sense” injury to the mother's heart and selectively home to theinjury zone. Secondly, there has been a great deal of controversy in thestem cell field whether stem cells other than embryonic stem (ES) cells,can give rise to functional, beating cardiomyocytes. Live imaging (datanot shown) by the present inventors using the new model described hereindemonstrated that this is possible. Thirdly, Cdx2 cells can home to theinjured heart and, in some embodiments, participate in cardiacdifferentiation in this injury model.

The present inventors were inspired by clinical observations that womenwith peripartum cardiomyopathy enjoy a high rate (˜50%) of spontaneousrecovery. This prompted the inventors to consider whether there may be afetal or placental contribution to maternal cardiac repair. Although thenew mouse injury model presented herein cannot precisely representperipartum cardiomyopathy, it is a model of fetal maternal cell transferwhich is believed to have identified appropriate cell types for cardiacregeneration. Briefly, mid-gestation myocardial infarction was inducedin pregnant female mice and they were sacrificed at various time points.Cells of fetal origin, marked by green fluorescent protein, homed to theinjured areas of the heart, but not to non-injured areas. They did nothome to non-injured organs within the mouse either, and this suggeststhat precise signals are ‘sensed’ by the fetal cells which enable themto target diseased tissue specifically. Upon homing to the heart, theydifferentiated into diverse cardiac lineages, including endothelialcells, smooth muscle cells, and cardiomyocytes. In vitro analysis offetal cells isolated from maternal hearts demonstrated that they canrecapitulate these differentiation pathways, forming vascular tubes in a3D collagen matrix and spontaneously beating cardiomyocytes whenco-cultured with neonatal cardiomyocytes. Although fetal cells isolatedfrom maternal heart express a variety of pluripotency markers, a notablenew finding was the finding that ˜40% expressed Caudal-related homeobox2(Cdx2), previously associated with trophoblast stem (TS) cells and otheraspects of non-cardiac development. This knowledge will spur furtherinvestigations into a potential role for TS cells in cardiacregeneration and further studies of the signaling mechanisms of cellsthat ‘naturally’ home to the diseased heart.

With regards to impact, the data presented herein implicate isolatingCdx2 cells from placenta for therapeutic use in heart disease. Thisrepresents a significant advance in the field because isolation of cellsfrom placenta avoids ethical concerns associated with ES cells asplacenta is routinely discarded in labor and delivery rooms throughoutthe world. These concepts have far-reaching applications in the fieldsof stem cell research and regenerative therapy.

Microchimerism results when two genetically disparate populations ofcells appear in the same tissue, organ, or individual. This can be dueto transfusion of blood products, organ transplantation, or pregnancy.In this study, we refer to microchimerism derived from the bidirectionaltrafficking and stable long-term persistence of allogeneic fetal cellsin the maternal host, a phenomenon that is common to many Eutheria.Microchimeric cells can modify immunological recognition or tolerance,affect the course and outcome of various diseases, and demonstrate stemcell-like or regenerative properties.

Fetal-maternal transfer of nucleated cells during pregnancy involvesmultiple cell types, some possessing multi-lineage potential and thesecells may appear transiently or persist for decades after delivery insome women The long-term survival of fetal CD34+ hematopoieticstem/progenitor cells, CD34+ and CD38+ lymphoid progenitors, CD3+ andCD14+ mononuclear cells, CD19+ and IgM+B lymphocyte precursor cells,CD45+ cells, desmin+ and mesenchymal stem cells have been reported inmaternal blood and tissues (Bianchi et al., 1996; Campagnoli et al.,2001; Fujiki et al., 2009; Khosrotehrani et al., 2008; Mikhail et al.,2008; Nguyen Huu et al., 2006; O'Donoghue et al., 2003; Osada et al.,2001). The rodent brain contains fetal chimeric progenitor cells (Tan etal., 2005) and fetal cells with regenerative potential have been foundin brain, liver, kidney, and lung injuries (Chen et al., 2001;Kleeberger et al., 2003; Wang et al., 2004). Fetal cells have also beenfound to participate in maternal neoangiogenesis during pregnancy atsites of skin inflammation (Nguyen Huu et al., 2007).

To the best of our knowledge, the phenomenon of fetal maternal stem celltransfer has never been explored in the realm of acute cardiac disease.One group has reported that cells of male fetus origin could be found inexplanted hearts of two women with idiopathic dilated cardiomyopathymany years after a previous pregnancy (Bayes-Genis et al., 2005). Thisobservational study did not determine whether the fetal cellscontributed to the development of cardiomyopathy or if their presencerepresented an attempt at cardiac regeneration.

These clinical observations led the present inventors to consider thatfetal or placental cells that enter the maternal circulation may berecruited to the sites of myocardial disease or injury to assist inrepair. Further, identification of the cell types implicated in thisprocess could be used for development of novel cell therapies for abroader spectrum of cardiovascular disease states. Significantcontroversy exists in the field of stem cell biology as to whether avariety of stem cell types other than embryonic stem (ES) cells can giverise to beating cardiomyocytes. The present studies illustrate for thefirst time that experimental myocardial injury, induced in a pregnantmouse, triggers the flux of fetal cells via the maternal circulationinto the injured heart where they undergo differentiation into diversecardiac cell fates. Fetal cells isolated from the maternal heart undergoclonal expansion and can differentiate into beating cardiomyocytes invitro. A significant proportion of the fetal cells homing to the heartexpress Cdx2, thus, trophoblast stem cells may participate in organrepair after acute injury.

Fetal-maternal transfer of nucleated cells during pregnancy involvesmultiple cell types, some possessing multi-lineage potential, and thesecells appear transiently or may persist for decades after delivery insome women. The long-term survival of fetal CD34+ hematopoieticstem/progenitor cells, CD34+ and CD38+ lymphoid progenitors, CD3+ andCD14+ mononuclear cells, CD19+ and IgM+B lymphocyte precursor cells,CD45+ cells, desmin+ and mesenchymal stem cells have been reported inmaternal blood and tissues. Fetal chimeric progenitor cells have beenfound in rodent brain and additionally, fetal cells with regenerativepotential have been found in brain, liver, kidney, and lung injuries.Fetal cells have also been found to participate in maternalneoangiogenesis during pregnancy at sites of skin inflammation.

The selective homing of eGFP+ cells in the model presented herein to thesite of maternal cardiac injury with lack of such homing to non-injuredtissues points to the presence of precise signals sensed by cells offetal origin that enable them to target diseased myocardiumspecifically, and to differentiate into diverse cardiac lineages (FIG.5A). Most notable is their differentiation into functionalcardiomyocytes that are able to beat in syncytium with neighboringcardiomyocytes (movie not shown), thus uncovering an evolutionarymechanism whereby a fetus may assist in protecting the mother's heartduring and after pregnancy.

This observation led the present inventors to consider that there may bea fetal or placental contribution to counteract maternal cardiac injury.While the mouse injury model presented herein is not an absolutelyprecise representation of peripartum cardiomyopathy, it does provide asound model system of murine fetomaternal microchimerism to identifyappropriate cell types for cardiac regeneration.

To this end, a far greater spectrum of potential applications to thefield of heart disease emerges from these studies. Prior to the presentwork described herein, the consensus in the field of cardiacregenerative medicine was that the ability of bone marrow-derived stemcells to differentiate into cardiomyocytes was questionable.

Several groups have demonstrated that ES cells (Nussbaum et al., 2007;van Laake et al., 2007; Xu et al., 2002; Yang et al., 2008) andendogenous populations of cardiac stem cells (Beltrami et al., 2003;Laugwitz et al., 2005a; Martin et al., 2004; Oh et al., 2003; Wu et al.,2008) have replicative and potentially regenerative capacities.

Notably, however, despite promising results with ES cells, there areethical issues regarding the use of embryonic material as well as thetendency of ES cells to form teratomas (Nussbaum et al., 2007). Nativecardiac progenitors, left in their natural milieu at their naturallyoccurring frequency, are clearly inadequate in reversing the downwardspiral of events culminating in heart failure. Many of these progenitortypes have not been reported to differentiate to functional beatingcardiomyocytes when tested ex vivo.

In contrast, utilizing live imaging, the present inventors demonstratedherein for the first time that fetal cells differentiate intospontaneously beating cardiomyocytes after homing to the heart.

The identification of Cdx2 by the present inventors as a unique andhighly prevalent marker expressed on fetal cells in the maternalmyocardium offers a new perspective regarding the appropriate cell typeto achieve these aims. The Cdx family of transcription factors consistof three mouse homologues (Cdx 1, 2, and 4) of the Drosophila caudalhomeobox genes, which are involved in specifying cell position along theanteroposterior axis, with similar functions in the later developmentalstages of the mouse embryo (Chawengsaksophak et al., 2004; Strumpf etal., 2005) as well as morphological specification of murine gut endoderm(Beck and Stringer, 2010; Chawengsaksophak et al., 1997). Cdx2 is alsorequired for trophectoderm fate commitment in the developing blastocyst(Niwa et al., 2005; Ralston and Rossant, 2005; Strumpf et al., 2005)(FIG. 5B). The trophectoderm gives rise to the trophoblast stem cellswhich have previously been associated solely with differentiation to theplacenta lineage (Ralston et al., 2010; Tanaka et al., 1998).

Bianchi and colleagues found that fetal cells that traffic to maternalblood and organs comprise a mixed population of progenitor anddifferentiated cells, with different relative proportions in differentmaternal organs (Fujiki et al., 2009) in a study that was performed inthe non-injured state. The results presented herein point towards thetransfer of several populations of progenitor cells. The present findingof Cdx2 cells of fetal or placental origin in the heart presents a celltype that is capable of cardiac differentiation under injury conditionsthat can be readily isolated from placenta.

Microchimerism results when two genetically disparate populations ofcells appear in the same tissue, organ, or individual. This can be dueto transfusion of blood products, organ transplantation, or pregnancy.As used herein, the term “microchimerism” refers to bidirectionaltrafficking and stable long-term persistence of allogeneic fetal cellsin the maternal host. Microchimeric cells can modify immunologicalrecognition or tolerance, affect the course and outcome of variousdiseases, and demonstrate stem cell-like or regenerative properties.

As use herein, the term “stem cell” refers to an undifferentiated,multipotent, self-renewing, cell. A stem cell is able to divide and,under appropriate conditions, has self-renewal capability and caninclude in its progeny daughter cells that can terminally differentiateinto any of a variety of different cell types. Hence, the stem cell is“multipotent” because stem cell progeny have multiple differentiationpathways. A stem cell is capable of self maintenance, meaning that witheach cell division, one daughter cell will also be on average a stemcell.

Non-stem cell progeny of a stem cell are typically referred to as“progenitor” cells, which are capable of giving rise to various celltypes within one or more lineages. As used herein, the term “progenitorcell” refers to an undifferentiated cell derived from a stem cell, andis not itself a stem cell. Some progenitor cells can produce progenythat are capable of differentiating into more than one cell type. Adistinguishing feature of a progenitor cell is that, unlike a stem cell,it does not exhibit self maintenance, and typically is thought to becommitted to a particular path of differentiation and will, underappropriate conditions, eventually differentiate along this pathway.

Stem cells and progenitor cells derived from a particular tissue arereferred to herein by reference to the tissue from which they wereobtained. For example, stem cells and progenitor cells obtained fromfetal tissue are referred to as “fetal stem cells” and “fetal progenitorcells,” respectively. Fetal tissue includes, but is not limited to,placenta used to feed a fetus during pregnancy, and which is expelledfollowing birth.

A “clonogenic population” refers to a population of cells derived fromthe same stem cell. A clonogenic population may include stem cells,progenitor cells, precursor cells, differentiated cells, or anycombination thereof.

The terms, “isolated,” “purified” and “enriched” indicate that the cellsare removed from their normal tissue environment and are present at ahigher concentration as compared to the normal tissue environment.Accordingly, an “isolated,” “purified” or “enriched” cell population mayfurther include cell types in addition to stem cells and/or progenitorcells and may include additional tissue components, and the terms“isolated,” “purified” and “enriched” do not necessarily indicate thepresence of only stem cells and progenitor cells. In one embodiment, an“isolated,” “purified” or “enriched” cell population contains greaterthan about 75% of the stem cells and/or progenitor cells. For example,an “isolated,” “purified” or “enriched” cell population contains greaterthan about 75%, about 80%, about 85%, about 90%, about 95%, about 97%,about 98% or more of the stem cells and/or progenitor cells.

Isolated tissue samples may be placed into culture without furtherprocessing, or they may be processed to release cells from other tissuecomponents by any of a variety of different means or combinationsthereof known in the art. Tissue may be physically processed, e.g., bycutting or mincing a tissue sample into smaller pieces. Cutting may beperformed by any conventional means available, including, e.g., the useof scissors, scalpels, razor blades, needles, and other sharpinstruments.

Tissue samples may be cultured in any of a variety of culture mediacapable of supporting cell viability, growth and/or attachment, such asserum-supplemented DMEM. In one embodiment, explant media (Iscove'sModified Dulbecco's IMDM with 10% fetal calf serum (FBS), 100 U/mlpenicillin G, 100 μg/ml streptomycin, 2 mmol/L L-glutamine, and 01mmol/L beta-mercaptoethanol) is used. Tissue samples may be culturedunder standard environmental conditions such as 37° C. and 5% CO₂.Tissue samples may be cultured for a time sufficient for adherent cellsto adhere and stem cells to migrate above the adherent cell layer, whichmay be, e.g., approximately one week, two weeks, three weeks or more.Generally, the age of donor tissue determines the time for culture: theolder the tissue, the longer the time it takes for the stem cells tomigrate out from the explant. Non-limiting representative examples oftissue and cell culture techniques useful for the present compositionsand methods of use thereof are provided in the Examples below.

Tissue may also be processed by exposure to an enzyme preparation thatfacilitates the release of cells from other tissue components. Examplesof such enzymes include, but are not limited to, matrixmetalloproteinases, pronase, clostripain, trypsin-like, pepsin-like,neutral protease-type and collagenases. Suitable proteolytic enzymes arecommercially available and are also described, for example, in U.S. Pat.Nos. 5,079,160; 6,589,728; 5,422,261; 5,424,208; and 5,322,790. In oneembodiment, the enzyme preparation is a collagenase preparation orcomprises collagenase. In other embodiments, the enzyme preparationcomprises one or more of trypsin-like, pepsin-like, clostripain, andneutral protease-type enzymes. For example, one suitable enzymepreparation may include a mixture of 0.2% trypsin and 0.1% collagenaseIV.

Stem cells and progenitor cells may be purified from other tissuecomponents after, or concurrent with, the processing of a tissue sample.In one embodiment, stem cells and progenitor cells may be purified fromother cells and tissue components after the tissue sample has beencultured under conditions suitable for cell growth and for a timesufficient to allow cells to adhere to the culture dish. Purification ofcells may include, for example, obtaining cells that migrate from thetissue sample during culture and may be present in the culture media orloosely adhered to the adherent fibroblast layer. The cells may beobtained by routine methods, such as removing and centrifuging the mediato pellet cells therein, and washing the cells remaining in the culturedish with a solution such as phosphate-buffered saline (PBS) or D-Hanksto remove those cells loosely attached to the adherent cell layer. Thiswash solution may then also be centrifuged to obtain cells.

Following isolation, a purified cell population may include at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% of Cdx-2 bearingstem cells or progenitor cells. The cells may also, in some embodiments,be characterized by the presence of one or more of the following cellmarkers: CD31, Sca-1, c-Kit, Pou5F1, Nanog, Isil, Sox2, Nkx2.5, CD23 andCdx2.

Cdx2 cells obtained from fetal placenta have been found by the presentinventors to have stem cell characteristics as they contribute tomultiple cell lineages. Cdx2 cells have been newly identified asprogenitors for endothelial cells, smooth muscle cellsand/cardiomyocytes. Provided herein is a composition comprising Cdx2cells or and one or more pharmaceutically acceptable carriers and/oradjuvants. The composition for delivery of cells includes the cells andcan comprise a pharmaceutical carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., buffered saline and thelike. These solutions are sterile and generally free of undesirablematter. These compositions can be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, albumin,anticoagulants such as CPD (citrate, phosphate, and dextrose), dextran,DMSO, combinations thereof, and the like. Biologically compatiblecarriers or excipients also include, but are not limited to, such as5-azacytidine, cardiogenol C, or ascorbic acid. The concentration ofactive agent in these formulations can vary widely, and can be selectedprimarily based on fluid volumes, viscosities, body weight, and thelike, in accordance with the particular mode of administration selectedand the subject's needs.

In one aspect, purified cell populations are present within acomposition adapted for, or suitable for, freezing and/or storage. Forexample, such a composition may further comprise fetal bovine serumand/or dimethylsulfoxide (DMSO).

In some embodiments, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the cells in a cell population described herein have thecapacity to undergo differentiation into specialized cell types. Forexample, the cells are capable of forming endothelial cells, smoothmuscle cells and/or cardiomyocytes.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for the preparation of amedicament for inducing cardiac regeneration.

In one embodiment, the composition increases cardiomyocyte formation,increase cardiomyocyte proliferation, increase cardiomyocyte cell cycleactivation, increase mitotic index of cardiomyocytes, increasemyofilament density, increase borderzone wall thickness, or acombination thereof, when administered to a subject.

In another embodiment, the composition treats myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy when administered to a subject.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for the preparation of amedicament for increasing cardiomyocyte formation, increasecardiomyocyte proliferation, increase cardiomyocyte cell cycleactivation, increase mitotic index of cardiomyocytes, increasemyofilament density, increase borderzone wall thickness, or acombination thereof.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for the preparation of amedicament for treating myocardial infarction, chronic coronaryischemia, arteriosclerosis, congestive heart failure, dilatedcardiomyopathy, restenosis, coronary artery disease, heart failure,arrhythmia, angina, atherosclerosis, hypertension, or myocardialhypertrophy.

Provided herein is a composition including a population of Cdx2 cellsand a pharmaceutically acceptable carrier for inducing cardiacregeneration.

In one embodiment, the composition increases cardiomyocyte formation,increase cardiomyocyte proliferation, increase cardiomyocyte cell cycleactivation, increase mitotic index of cardiomyocytes, increasemyofilament density, increase borderzone wall thickness, or acombination thereof, when administered to a subject.

In another embodiment, the composition treats myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy when administered to a subject.

A composition including a population of Cdx2 cells and apharmaceutically acceptable carrier for increasing cardiomyocyteformation, increase cardiomyocyte proliferation, increase cardiomyocytecell cycle activation, increase mitotic index of cardiomyocytes,increase myofilament density, increase borderzone wall thickness, or acombination thereof.

A composition including a population of Cdx2 cells and apharmaceutically acceptable carrier for treating myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy.

Provided herein is a composition comprising a population of cells and apharmaceutically acceptable carrier for increasing cardiomyocyteformation, increase cardiomyocyte proliferation, increase cardiomyocytecell cycle activation, increase mitotic index of cardiomyocytes,increase myofilament density, increase borderzone wall thickness, or acombination thereof, wherein said cells express one or more markersidentified in Table 2 or in FIG. 5C. In one embodiment, the cells arederived from placenta. In another embodiment, the cells are progenitorcells or stem cells. In another embodiment, the cells express Cdx2, Cd9,Eomes, CD34, CD31, c-kit or a combination thereof. In anotherembodiment, the cells express Cdx2 and Cd9.

Also provided herein is a composition comprising a population of cellsand a pharmaceutically acceptable carrier for treating myocardialinfarction, chronic coronary ischemia, arteriosclerosis, congestiveheart failure, dilated cardiomyopathy, restenosis, coronary arterydisease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, or myocardial hypertrophy, wherein said cells express oneor more markers identified in Table 2 or in FIG. 5C. In one embodiment,the cells are derived from placenta. In another embodiment, the cellsare progenitor cells or stem cells. In another embodiment, the cellsexpress Cdx2, Cd9, Eomes, CD34, CD31, c-kit or a combination thereof. Inanother embodiment, the cells express Cdx2 and Cd9.

Also provided herein is a composition comprising a population of cellsand a pharmaceutically acceptable carrier for inducing cardiacregeneration, wherein said cells express one or more markers identifiedin Table 2 or in FIG. 5C. In one embodiment, the cells are derived fromplacenta. In another embodiment, the cells are progenitor cells or stemcells. In another embodiment, the cells express Cdx2, Cd9, Eomes, CD34,CD31, c-kit or a combination thereof. In another embodiment, the cellsexpress Cdx2 and Cd9. In another embodiment, the composition increasescardiomyocyte formation, increase cardiomyocyte proliferation, increasecardiomyocyte cell cycle activation, increase mitotic index ofcardiomyocytes, increase myofilament density, increase borderzone wallthickness, or a combination thereof, when administered to a subject. Inanother embodiment, the composition treats myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy when administered to a subject.

Provided herein is a composition comprising a population of cells and apharmaceutically acceptable carrier for increasing cardiomyocyteformation, increase cardiomyocyte proliferation, increase cardiomyocytecell cycle activation, increase mitotic index of cardiomyocytes,increase myofilament density, increase borderzone wall thickness, or acombination thereof, wherein said cells express one or more markersidentified in Table 2 or in FIG. 5C. In one embodiment, the cells arederived from placenta. In another embodiment, the cells are progenitorcells or stem cells. In another embodiment, the cells express Cdx2, Cd9,Eomes, CD34, CD31, c-kit or a combination thereof. In anotherembodiment, the cells express Cdx2 and Cd9.

Also provided herein is a composition comprising a population of cellsand a pharmaceutically acceptable carrier for treating myocardialinfarction, chronic coronary ischemia, arteriosclerosis, congestiveheart failure, dilated cardiomyopathy, restenosis, coronary arterydisease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, or myocardial hypertrophy, wherein said cells express oneor more markers identified in Table 2 or in FIG. 5C. In one embodiment,the cells are derived from placenta. In another embodiment, the cellsare progenitor cells or stem cells. In another embodiment, the cellsexpress Cdx2, Cd9, Eomes, CD34, CD31, c-kit, or a combination thereof.In another embodiment, the cells express Cdx2 and Cd9.

Also provided herein is a composition comprising a population of cellsand a pharmaceutically acceptable carrier for inducing cardiacregeneration, wherein said cells express one or more markers identifiedin Table 2 or in FIG. 5C. In one embodiment, the cells are derived fromplacenta. In another embodiment, the cells are progenitor cells or stemcells. In another embodiment, the cells express Cdx2, Cd9, Eomes, CD34,CD31, c-kit or a combination thereof. In another embodiment, the cellsexpress Cdx2 and Cd9. In another embodiment, the composition increasescardiomyocyte formation, increase cardiomyocyte proliferation, increasecardiomyocyte cell cycle activation, increase mitotic index ofcardiomyocytes, increase myofilament density, increase borderzone wallthickness, or a combination thereof, when administered to a subject. Inanother embodiment, the composition treats myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy when administered to a subject.

One would understand that one or more additional excipients, carriers,adjuvants, cells, etc., may be added to the compositions describedherein.

Compositions described herein may contain, for example, from about 1×10⁸to about 1×10² cells. Compositions may also contain, for example, fromabout 1×10⁶ to about 1×10⁵ cells. In one embodiment where additionalcells are present in the composition, the amount of cells in thecomposition will generally contain about 1×10⁸ to about 1×10² Cdx2cells. In another embodiment, where additional cells are present in thecomposition, the composition will generally contain about 1×10⁶ to about1×10⁵ Cdx2 cells.

One would understand that the amount of cells to be formulated in acomposition or medicament for administration to a subject will dependupon the subject to be treated and the optimal dose or doses (in thecase of repeat therapy) can be empirically determined by the treatingdoctor. For example, height, weight, age, gender, and overall physicalcondition may be considered by a doctor in determining a therapeuticallyeffective amount of cells to administer. A therapeutically effectiveamount of cells is one which is capable of partially or fully restoringcardiac function and/or treating a heart condition. In one embodiment,the amount of composition comprises about from 1×10⁸ to about 1×10²cells. In another embodiment, the amount of introduced compositioncomprises from about 1×10⁶ to about 1×10⁵ cells.

Given the pervasiveness of cardiac disease, there exists a need fortherapeutic cell replacement strategies utilizing transplantation ofautologous and/or exogenous cells for the treatment or prevention ofheart disease. There also exists a need for mouse models that mimicmyocardial infarction and which can be used to study the effect of celltransplantation therapies.

The present inventors determined for the first time that fetal cellsselectively home (migrate) to injured maternal hearts and undergodifferentiation into diverse cardiac lineages. Utilizing enhanced greenfluorescent protein (eGFP) tagged fetuses, engraftment of multipotentfetal cells in injury zones of maternal hearts was demonstrated.

In vivo, eGFP+ fetal cells were found to form endothelial cells, smoothmuscle cells, and cardiomyocytes. In vitro, fetal cells isolated frommaternal hearts recapitulate these differentiation pathways,additionally forming vascular tubes and beating cardiomyocytes in afusion-independent manner. Fetal maternal stem cell transfer was foundto have an effect on maternal response to cardiac injury. Furthermore,Cdx2 cells were identified as a novel cell type for cardiovascularregenerative therapy.

The results of the studies described herein represent a significantadvance in the field because isolation of cells from placenta avoidsethical concerns associated with ES cells as placenta is routinelydiscarded in labor and delivery rooms throughout the world. Theseconcepts have far-reaching applications in the fields of stem cellresearch and regenerative therapy.

Provided herein is an isolated population of Cdx2 stem cells use in acomposition or medicament for prevention or treatment of myocardialinfarction, chronic coronary ischemia, arteriosclerosis, congestiveheart failure, dilated cardiomyopathy, restenosis, coronary arterydisease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, or myocardial hypertrophy. In one embodiment, the Cdx-2bearing stem cells are derived from placenta. Provided herein is anisolated population of Cdx2 cells capable of restoring heart functionand of forming endothelial cells, smooth muscle cells and/orcardiomyocytes. The approaches described herein are based, in part, uponapplication of the discovery of the ability of Cdx2 cells to home to thematernal heart and treat myocardia/infarction in the model provided inthe Examples below.

The present cells provide a novel cellular therapeutic agent for tissuerepair. Such therapeutic tissue repair utilizes Cdx2 cells, which may beisolated from placental tissue and can be transplanted in an autologousmanner. Methods and compositions described herein can be directed to,for example, cardiac repair.

Cdx2 cells introduced into the peri-infarct zone of infarcted mousehearts can form endothelial cells, smooth muscle cells and/orcardiomyocytes and may induce myocardial repair, prevent heart failure,and induce cardiac remodeling. Thus, Cdx2 cells obtained from, forexample, placental tissue, may be administered to a patient with, or atrisk for, myocardial infarction, chronic coronary ischemia,arteriosclerosis, congestive heart failure, dilated cardiomyopathy,restenosis, coronary artery disease, heart failure, arrhythmia, angina,atherosclerosis, hypertension, or myocardial hypertrophy.

Fetal stem cells naturally home to sites of maternal cardiac injuryduring pregnancy. The fetal cells are capable of differentiating intodiverse cardiac lineages in vivo, including endothelial and smoothmuscle cells and cardiomyocytes. They recapitulate these differentiationpathways in vitro, forming vascular tubes and spontaneously beatingcardiomyocytes.

Cdx2 has been identified herein as a unique and highly prevalent markerexpressed in fetal cells isolated from maternal myocardium, offering anew perspective regarding the appropriate cell type best suited forcardiovascular cell therapy. Cdx2 is required for trophectoderm fatecommitment in the developing blastocyst. The trophectoderm gives rise tothe trophoblast stem (TS) cells which have previously been associatedsolely with differentiation to the placenta lineage. Cdx2 cells may beisolated from end-gestation placentas and be utilized for allogeneicstem cell transplantation. These studies may be used for clinicaltesting and use of placenta-derived Cdx2 cells in the treatment of heartdisease.

The present inventor is the first to propose isolation of Cdx2 cells anda heterogeneous mix of fetal-derived placenta cells from end-gestationmouse and human placentas, testing their differentiation properties invitro, and identifying cell surface markers that may be used tofacilitate further sorting.

A lentivirus has been constructed in which the murine Cdx2 promoterdrives expression of the reporter gene tdTomato. The control lentivirusemploys a cytomegalovirus promoter driving tdTomato. Cdx2 cells areisolated from murine and human placentas based on the red fluorescenceof tdTomato. Single cell sorting into 96-well plates is performed toconfirm clonality. These cells are then cultured on cardiac mesenchymalfibroblasts and neonatal cardiomyocytes to test for their ability todifferentiate into endothelial cells, smooth muscle cells, andcardiomyocytes. Live-imaging microscopy is utilized to assessspontaneous beating of Cdx2 cell-derived cardiomyocytes. A heterogeneousmix of fetal-derived placenta cells that are mononuclear will also betested in 96-well plates for clonality and in cell culture to examinetheir differentiation pathways. Murine fetal cells will be isolatedusing green-fluorescent protein (GFP) after wild-type virgin female miceare mated with transgenic GFP mice as described in the examples.Fetal-derived cells will also be isolated from human placentas byseparating the fetal portion of placentas of first-time mothers who havegiven birth to males according to established techniques and confirmingfetal identity with Y-chromosome FISH as described in the examples. Cdx2cells will be isolated from human placenta tissues and proteomicapproaches employed to search for novel cell surface markers that may beutilized for FACS sorting.

The present inventor also tested the ability of Cdx2 cells versus aheterogeneous mix of fetal-derived cells isolated from placenta to formcardiomyocytes and blood vessels in vivo and restore cardiac functionvia transplantation experiments in the post-myocardial infarctionsetting. Cdx2 cells versus heterogeneous fetal-derived placenta cells(hfpcs) cardiovascular differentiation potential in vivo and theirability to restore cardiac function in a rodent model.Immunohistochemical approaches will be utilized to detect formation ofendothelial cells, smooth muscle cells, and cardiomyocytes in theinfarcted hearts. Cardiac function enhancement will be detected withmagnetic resonance imaging (MRI).

The methods described herein involve intramyocardial transplantation ofCdx2 cells. Such therapeutic methods may repair and regenerate damagedmyocardium and restore cardiac function after, for example, acutemyocardial infarction and/or other ischemic or reperfusion relatedinjuries. Methods generally include contacting a composition containingCdx2 cells with cardiac tissue or cells. Contacting may occur viainjection methods known in the art and described herein.

Provided herein is a method for restoring cardiac function comprisingintroducing an effective amount of a composition Cdx2 cells and apharmaceutically acceptable carrier into a heart of a subject in needthereof. Restoration of cardiac function may include partial or completerestoration. In one embodiment, at least 50% of cardiac function isrestored compared to a patient who does not receive such treatment. Inanother embodiment, about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of cardiac function is restored. A subject receiving treatment mayalso be tested in various ways for cardiac health and have an improvedresult observed by echocardiography, multi-gated acquisition scan (MUGA)scan, nuclear stress test, radionuclide angiography, left ventricularangiography, MRI or ECG. In one embodiment, a patient's cardiac functiondoes not worsen.

Provided herein is a method of inducing cardiomyocyte regeneration,cardiac repair, vasculogenesis or cardiomyocyte differentiation,including contacting a composition comprising Cdx2 cells with injuredheart tissue. In one embodiment, Cdx2 cells are fetal stem cells whichmay be derived from placenta. The Cdx2 cells may be substantiallyisolated cells. In one embodiment, the Cdx2 cells represent at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100% ofthe cells in the composition.

In such methods, a subject may be diagnosed with, or at risk for,myocardial infarction, chronic coronary ischemia, arteriosclerosis,congestive heart failure, dilated cardiomyopathy, restenosis, coronaryartery disease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, or myocardial hypertrophy. In one embodiment, the subjectis diagnosed with myocardial infarction.

In another embodiment, the subject has, or is at risk for, heartfailure.

Where compositions such as those described herein are utilized fortreatment of a subject, introducing or contacting the composition withthe heart of the subject can occur by implanting the composition intocardiac tissue of the subject. Alternatively, introducing or contactingthe composition can occur via injecting the composition into the subjectusing conventional techniques in the art. Cardiac tissue to be treatedaccording to the present methods includes, for example, myocardium,endocardium, epicardium, connective tissue in the heart, and nervoustissue in the heart. Animals such as mammals represent subjects to betreated with the presently disclosed compositions and methods. In oneembodiment, the subject is a human, a veterinary animal, a primate, adomesticated animal, a reptile, or an avian. For example, a humansubject may be treated with the disclosed compositions to restorecardiac function and to treat one or more heart-related conditions.

Provided herein is a method of preventing or treating a patientsuffering from myocardial infarction, chronic coronary ischemia,arteriosclerosis, congestive heart failure, dilated cardiomyopathy,restenosis, coronary artery disease, heart failure, arrhythmia, angina,atherosclerosis, hypertension, or myocardial hypertrophy comprisingadministering a composition comprising an isolated stem cell populationcomprising Cdx2 cells and a pharmaceutically acceptable carrier.

Another aspect provided herein is a method for restoring cardiacfunction comprising introducing an effective amount of a compositioncells and a pharmaceutically acceptable carrier into a heart of asubject in need thereof, wherein said cells express one or more markersidentified in Table 2 or in FIG. 5C. Also provided herein is a method ofinducing cardiomyocyte regeneration, cardiac repair, vasculogenesis orcardiomyocyte differentiation, comprising contacting cells with injuredheart tissue, wherein said cells express one or more markers identifiedin Table 2 or in FIG. 5C.

In one embodiment, the cells are derived from placenta. In anotherembodiment, the cells are progenitor cells or stem cells. In anotherembodiment, the cells express Cdx2, Cd9, Eomes, CD34, CD31, c-kit, or acombination thereof. In another embodiment, the cells express Cdx2 andCd9.

Generally, a subject upon which the methods of the invention are to beperformed will have been diagnosed with myocardial infarction, chroniccoronary ischemia, arteriosclerosis, congestive heart failure, dilatedcardiomyopathy, restenosis, coronary artery disease, heart failure,arrhythmia, angina, atherosclerosis, hypertension, or myocardialhypertrophy. Alternatively, it will have been determined that a subjectupon which the methods of the invention are performed is at risk formyocardial infarction, chronic coronary ischemia, arteriosclerosis,congestive heart failure, dilated cardiomyopathy, restenosis, coronaryartery disease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, or myocardial hypertrophy based on assessment of the hearttissue and/or family history. In one embodiment, a subject has beendiagnosed with myocardial infarction or at risk for heart failure.

One in this field of endeavor would understand that methods ofprevention are intended for subjects that have a family history of heartattacks or may physically be predisposed to heart attacks. Thus,prevention encompasses administration of compositions described hereinto a subject to prevent damage to the subject's heart and/or to preventacute myocardial infarction. A subject that has been treated with suchmethods may experience an overall improvement in health. Additionally,cardiac function may be restored and/or improved as described abovecompared to lack of treatment.

In accordance with one embodiment, a composition containing Cdx2 cellsis introduced into the cardiac tissue or cells a subject. Briefly, thismethod may be performed as follows: Cdx2 cells can be isolated fromplacenta using conventional means known in the art and described herein.Once isolated, the stem cells can be purified and/or expanded. Theisolated cells can then be formulated as a composition (medicament)comprising the Cdx2 cells along with, for example, a pharmaceuticallyacceptable carrier. The composition (medicament) so formed can then beintroduced into the heart tissue of a subject.

A subject to be treated with the disclosed compositions and methods willhave been diagnosed as having, or being at risk for, a heart condition,disease, or disorder. Introduction of the composition can be accordingto methods described herein or known in the art. For example, the Cdx2cell composition can be administered to a subject's heart by way ofdirect injection delivery or catheter delivery. Introduction of Cdx2cells can be a single occurrence or can occur more than one time over aperiod of time selected by the attending physician.

The time course and number of occurrences of Cdx2 cell implantation intoa subject's heart can be dictated by monitoring generation and/orregeneration of cardiac tissue, where such methods of assessment anddevisement of treatment course is within the skill of the art of anattending physician.

Cardiac tissue into which Cdx2 cells can be introduced includes, but isnot limited to, the myocardium of the heart (including cardiac musclefibers, connective tissue (endomysium), nerve fibers, capillaries, andlymphatics); the endocardium of the heart (including endothelium,connective tissue, and fat cells); the epicardium of the heart(including fibroelastic connective tissue, blood vessels, lymphatics,nerve fibers, fat tissue, and a mesothelial membrane consisting ofsquamous epithelial cells); and any additional connective tissue(including the pericardium), blood vessels, lymphatics, fat cells,progenitor cells (e.g., side-population progenitor cells), and nervoustissue found in the heart. Cardiac muscle fibers are composed of chainsof contiguous heart-muscle cells (cardiomyocytes), joined end to end atintercalated disks. These disks possess two kinds of cell junctions:expanded desmosomes extending along their transverse portions, and gapjunctions, the largest of which lie along their longitudinal portions.Each of the above tissues can be selected as a target site forintroduction of Cdx2 cells, either individually or in combination withother tissues.

A determination of the need for treatment will typically be assessed bya history and physical exam consistent with the myocardial defect,disorder, or injury at issue. Subjects with an identified need oftherapy include those with diagnosed damaged or degenerated heart tissue(i.e., heart tissue which exhibits a pathological condition) or whichare predisposed to damaged or degenerative heart tissue. Causes of hearttissue damage and/or degeneration include, but are not limited to,chronic heart damage, chronic heart failure, damage resulting frominjury or trauma, damage resulting from a cardiotoxin, damage fromradiation or oxidative free radicals, damage resulting from decreasedblood flow, and myocardial infarction (such as a heart attack). In oneembodiment, a subject in need of treatment according to the methodsdescribed herein has been diagnosed with degenerated heart tissueresulting from a myocardial infarction or heart failure.

It should be recognized that methods disclosed herein can be practicedin conjunction with existing myocardial therapies to effectively treator prevent disease. The methods, compositions, and devices of theinvention can include concurrent or sequential treatment withnon-biologic and/or biologic drugs.

The subject receiving cardiac implantation of Cdx2 cells according tothe methods described herein will usually have been diagnosed as having,or being at risk for, a heart condition, disease, or disorder. Themethods of the invention can be useful to alleviate the symptoms of avariety of disorders, such as disorders associated with aberrantcell/tissue damage, ischemic disorders, and reperfusion relateddisorders. For example, the methods are useful in alleviating a symptomof myocardial infarction, chronic coronary ischemia, arteriosclerosis,congestive heart failure, dilated cardiomyopathy, restenosis, coronaryartery disease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, myocardial hypertrophy, or a combination thereof. Themethods of the invention can also be useful to prevent the symptoms of avariety of disorders, such as disorders associated with aberrantcell/tissue damage, ischemic disorders, and reperfusion relateddisorders. For example, the methods are useful in preventing a symptomof myocardial infarction, chronic coronary ischemia, arteriosclerosis,congestive heart failure, dilated cardiomyopathy, restenosis, coronaryartery disease, heart failure, arrhythmia, angina, atherosclerosis,hypertension, myocardial hypertrophy, or a combination thereof. Thecondition, disease, or disorder can be diagnosed and/or monitored,typically by a physician using standard methodologies.

Alleviation of one or more symptoms of the condition, disease, ordisorder indicates that the composition confers a clinical benefit, suchas a reduction in one or more of the following symptoms: shortness ofbreath, fluid retention, headaches, dizzy spells, chest pain, leftshoulder or arm pain, and ventricular dysfunction. One would understandthat a reduction of one more of the symptoms need not be 100% to providetherapeutic benefit to the subject being treated. Thus, in oneembodiment, a reduction of about 50%, about 60%, about 70%, about 80%,about 90%, or more of one or more such symptoms may provide sufficienttherapeutic relief to a patient.

With respect to methods of prevention, one would understand thatprevention does not necessarily mean that a patient never experiencescardiac damage. Rather, prevention includes, but is not limited to,delay of onset of one or more symptoms compared to a lack of treatment.In one non-limiting example, a patient who has a family history of fatalheart attacks by 50 years of age may experience one or more symptomsdescribed herein, but not experience a fatal heart attack or mayexperience a less severe heart attack compared to lack of treatment.

Cardiac cell/tissue damage is characterized, in part, by a loss of oneor more cellular functions characteristic of the cardiac cell type whichcan lead to eventual cell death. For example, cell damage to acardiomyocyte results in the loss of contractile function of the cellresulting in a loss of ventricular function of the heart tissue. Anischemic or reperfusion related injury results in tissue necrosis andscar formation. Injured myocardial tissue is defined for example bynecrosis, scarring, or yellow softening of the myocardial tissue.Injured myocardial tissue leads to one or more of several mechanicalcomplications of the heart, such as ventricular dysfunction, decreasedforward cardiac output, as well as inflammation of the lining around theheart (i.e., pericarditis). Accordingly, regenerating injured myocardialtissue according to the methods described herein can result inhistological and functional restoration of the tissue.

The methods described herein can promote generation and/or regenerationof heart tissue, and/or promote endogenous myocardial regeneration ofheart tissue in a subject. Promoting generation of heart tissuegenerally includes, but is not limited to, activating, enhancing,facilitating, increasing, inducing, initiating, or stimulating thegrowth and/or proliferation of heart tissue, as well as activating,enhancing, facilitating, increasing, inducing, initiating, orstimulating the differentiation, growth, and/or proliferation of hearttissue cells. Thus, the methods include, for example, initiation ofheart tissue generation, as well as facilitation or enhancement of hearttissue generation already in progress. Differentiation is generallyunderstood as the cellular process by which cells become structurallyand functionally specialized during development. Proliferation andgrowth, as used herein, generally refer to an increase in mass, volume,and/or thickness of heart tissue, as well as an increase in diameter,mass, or number of heart tissue cells. The term generation is understoodto include the generation of new heart tissue and the regeneration ofheart tissue where heart tissue previously existed.

Generation of new heart tissue and regeneration of heart tissue,resultant from the therapeutic methods described herein, can be detectedand/or measured using conventional procedures in the art. Suchprocedures include, but are not limited to, Western blotting forheart-specific proteins, electron microscopy in conjunction withmorphometry, simple assays to measure rate of cell proliferation(including trypan blue staining, the Cell Titer-Blue cell viabilityassay from Promega (Madison, Wis.), the MTT cell proliferation assayfrom American Type Culture Collection (ATCC), differential staining withfluorescein diacetate and ethidium bromide/propidium iodide, estimationof ATP levels, flow-cytometry assays, etc.), and any of the methods,molecular procedures, and assays disclosed herein.

Cdx2 cells can be isolated from placental tissue, purified, and culturedas described in the present examples. Additional art-recognized methodsof isolating, culturing, and differentiating stems cells are generallyknown in the art (see, e.g., Lanza et al., eds. (2004) Handbook of StemCells, Academic Press, ISBN 0124366430; Lanza et al., eds. (2005)Essentials of Stem Cell Biology, Academic Press, ISBN 0120884429;Saltzman (2004) Tissue Engineering: Engineering Principles for theDesign of Replacement Organs and Tissues, Oxford ISBN 019514130X;Vunjak-Novakovic and Freshney, eds. (2006) Culture of Cells for TissueEngineering, Wiley-Liss, ISBN 0471629359; Minuth et al. (2005) TissueEngineering: From Cell Biology to Artificial Organs, John Wiley & Sons,ISBN 3527311866). Such methods can be utilized directly or adapted foruse with the Cdx2 cells described herein.

It will be appreciated that the time between isolation, culture,expansion, and/or implantation may vary according to a particularapplication and/or a particular subject. Incubation (and subsequentreplication and/or differentiation) of a composition containing Cdx2cells can be, for example, at least in part in vitro, substantially invitro, at least in part in vivo, or substantially in vivo. Determinationof optimal culture time may be empirically determined.

Cdx2 cells can be derived from placenta of the same or different speciesas the transplant recipient. For example, progenitor cells can bederived from an animal, including but not limited to, mammals, reptiles,and avians such as, for example, horses, cows, dogs, cats, sheep, pigs,chickens, and humans. In one embodiment, Cdx2 cells are derived fromhuman placenta. It is also contemplated that autologous Cdx2 cells maybe obtained from the subject, into which the Cdx2 cells arere-introduced. Such autologous Cdx2 cells may be expanded and/ortransformed, as described herein, before re-introduction to the host.

Cdx2 cells can be obtained by screening a plurality of cells fromplacental tissue. After screening, Cdx2 cells may be selected andprepared for transplantation. In one aspect, therapeutic Cdx2 cells maybe expanded ex vivo (or in vitro) using, for example, standard methodsused to culture Cdx2 cells and maintain stable cell lines.Alternatively, these cells can be expanded in vivo (i.e., afterimplantation). These cells can also be used for future transplantationprocedures. The screened and isolated cells may, optionally, be furtherenriched for Cdx2 cells prior to transplantation. Methods to select forstem cells, for example Cdx2 cells, are well known in the art (e.g.,MoFlow Cell Sorter). For example, samples can be enriched by taggingcell-surface markers of undifferentiated Cdx2 cells with fluorescentlylabeled monoclonal antibodies and sorting via fluorescence-activatedcell sorting (FACS). Alternatively, a sample of the Cdx2 cell-richculture can be implanted without further enrichment.

Isolated Cdx2 cells can optionally be transformed with a heterologousnucleic acid so as to express a bioactive molecule or heterologousprotein or to overexpress an endogenous protein. Transformation of stemcells, including Cdx2 cells, may be conducted using conventional methodsin the art. In one non-limiting example, Cdx2 cells may be geneticallymodified to expresses a fluorescent protein marker (e.g., GFP, eGFP,BFP, CFP, YFP, RFP, etc.). Marker protein expression can be especiallyuseful in implantation scenarios, as described herein, so as to monitorCdx2 cell placement, retention, and replication in target tissue. Asanother example, Cdx2 cells may be transfected with one or more geneticsequences that are capable of reducing or eliminating an immune responsein the host (e.g., expression of cell surface antigens such as class Iand class II histocompatibility antigens may be suppressed). This mayallow the transplanted cells to have reduced chance of rejection by thehost, especially where the cells were from a different subject.

It may be desirable in some cases to increase levels of endogenous cellcycle regulators in Cdx2 cells and/or introduce exogenous cell cycleregulators into Cdx2 cells. For example, Cdx2 cells may be geneticallyengineered to express increased levels of cyclin A2 such that the cellshave augmented and/or prolonged proliferative potential. The Cdx2 cellsmay be contacted with, or transformed to express or overexpress, avariety of cell cycle regulators so as to achieve similar results.Elevated levels of an active cell cycle regulator (e.g., a cyclin) inCdx2 cells may be accomplished by, for example, contacting ortransforming the Cdx2 cells with a cell cycle regulator protein, or aprotein variant thereof, or a cell cycle regulator-associated agent.Cyclin proteins include, but are not necessarily limited to, cyclins A,B, C, D, and E. In one embodiment, the level of active cyclin A2 in theCdx2 cell is elevated (see, e.g., U.S. Publication No. 2006/0160733 A1,which is incorporated by reference herein). Various transport agents anddelivery systems may be employed so as to effect intracellular transportof the cyclin protein into Cdx2 cells (see, e.g., Stayton et al. (2005)Orthod. Craniofacial. Res., 8: 219-225). Isolated Cdx2 cells may betransduced with, for example, a lentiviral vector, retroviral vector,adenoviral vector, adeno-associated viral vector, or other vectorsystem, overexpressing a cyclin gene. Several ways are available forincreasing cyclin A2 expression including, but not limited to,transducing isolated Cdx2 cells with a lentiviral vector overexpressinga cyclin A2 gene, or providing cells with a nanoparticle that transferscyclin A2, a protein composition or a small molecule that activatescyclin A2 in a cell. Any other method for inducing cyclin A2 usingconventional means is also included herein.

In one embodiment, contact of Cdx2 cells with cyclin A2 may occurbefore, during, or after isolation and/or purification. Similarly,contact of Cdx2 cells with cyclin A2 may occur before, during, or afterimplantation into a subject. Cyclin A2 may be generated by synthesis ofpolypeptides in vitro, e.g., by chemical means, or in vitro translationof mRNA (see, e.g., U.S. Publication No. 2006/0160733). For example, acyclin A2 may be synthesized by conventional methods in the art (see,e.g., Benoiton (2005) Chemistry of Peptide Synthesis, CRC, ISBN1574444549; Goodman et al., eds. (2004) Synthesis Of Peptides AndPeptidomimetics: Workbench Edition, Thieme Medical Pub, ISBN1588903117).

Fetal Cdx2 cells may be cultured and/or implanted along with otherprogenitor cell types. For example, Cdx2 cells obtained from placentamay be cultured and/or implanted along with other stem cells, such asmesenchymal stem cells. Alternatively, or in addition, Cdx2 cells may becultured and/or implanted along with cardiomyocytes.

Provided herein are methods for enhancing cardiac function in a subjectin need thereof by introducing Cdx2 cells into the heart of a subject.Cdx2 cell compositions may be directly introduced into, or contactedwith, cardiac tissue and/or cells. Introduction to the tissues or cellsof a subject may occur ex vivo or in vivo. In one embodiment,compositions containing isolated cells are directly implanted intocardiac tissue of the subject, in vivo.

Therapeutic cells may be implanted into a subject using conventionalmethods (see, for example, the present Examples and Orlic et al. (2001)Nature, 410(6829): 701-705). For example, cells, or compositionscomprising cells, may be introduced via direct injection (e.g.,intermyocardial or intercoronary injection) or catheter-based delivery(e.g., intermyocardial, intercoronary, orcoronary sinus delivery).Intercoronary catheter delivery directly injects cells into hearttissue.

In one aspect, the cells may be transplanted along with a carriermaterial, such as collagen or fibrin glue or other scaffold materials.Such materials may improve cell retention and integration afterimplantation. Exemplary materials and methods for employing them areknown in the art and are contemplated herein (see, e.g., Saltzman (2004)Tissue Engineering: Engineering Principles for the Design of ReplacementOrgans and Tissues, Oxford ISBN 019514130X; Vunjak-Novakovic andFreshney, eds. (2006) Culture of Cells for Tissue Engineering,Wiley-Liss, ISBN 0471629359; and Minuth et al. (2005) TissueEngineering: From Cell Biology to Artificial Organs, John Wiley & Sons,ISBN 3527311866).

The amount of cells introduced into the heart tissue of the subject canbe that amount sufficient to improve cardiac function, increasecardiomyocyte formation, and/or increase mitotic index ofcardiomyocytes. For example, an effective amount may increasecardiomyocyte formation, increase cardiomyocyte proliferation, increasecardiomyocyte cell cycle activation, increased mitotic index ofcardiomyocytes, increase myofilament density, increase borderzone wallthickness, or a combination thereof. An effective amount may formendothelial cells, smooth muscle cells, cardiomyocytes, or a combinationthereof.

An effective amount of cells to be administered can be, for example,about 1×10⁸ to about 100 cells. For example, about 1×10⁸, about 1×10⁷,about 1×10⁶, about 1×10⁵, about 1×10⁴, about 1×10³, about 1×10² cellscan constitute an effective amount. In certain embodiments, about 1×10⁶to about 1×10⁵ cells are introduced. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific cells employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration; the route of administration;the duration of the treatment; drugs used in combination or coincidentalwith the specific composition employed and like factors well known inthe medical arts. In certain instances, the total desired effectiveamount may be divided into multiple doses for purposes ofadministration. Consequently, single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. It will beunderstood, however, that the total dosage of the compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment.

Improving or enhancing cardiac function generally refers to improving,enhancing, augmenting, facilitating or increasing the performance,operation, or function of the heart and/or circulatory system of asubject. Improving or enhancing cardiac function may also refer to animprovement in one or more of the following symptoms: chest pain(typically radiating to the left arm or left side of the neck),shortness of breath, nausea, vomiting, palpitations, sweating, andanxiety. The amount of cells introduced into the heart tissue of thesubject can be that amount sufficient to forming endothelial cells,smooth muscle cells and/cardiomyocytes. An improvement in cardiacfunction may be readily assessed and determined based on knownprocedures including, but limited to, an electrocardiogram (ECG),echocardiography, measuring volumetric ejection fraction using magneticresonance imaging (MRI) and/or one or more blood tests. The most oftenused markers for blood tests are the creatine kinase-MB (CK-MB) fractionand the troponin levels.

Introduction of cell-containing compositions can occur as a single eventor over a time course of treatment. For example, compositions can beadministered daily, weekly, bi-weekly, or monthly. For treatment ofacute conditions, the time course of treatment generally will be atleast several days. Certain conditions may extend treatment from severaldays to several weeks. For example, treatment could extend over oneweek, two weeks, or three weeks. For chronic conditions or preventativetreatments, treatment regimens may extend from several weeks to severalmonths or even a year or more.

Provided herein is a mouse model to study myocardial infarction and therole of fetal cells in treatment of cardiac injury. The model comprises(1) mating wild-type female mice with eGFP positive male mice to formeGFP positive fetuses; (2) inducing myocardial infarction in thepregnant mice at E12 days; (3) assessing maternal hearts for eGFPpositive cells, wherein the presence of eGFP positive cells indicatesmigration of fetal cells to the material heart and/or assessing one ormore symptoms of myocardial infarction. In one embodiment eGFP positivecells have differentiated and have formed endothelial cells, smoothmuscle cells and/or cardiomyocytes.

In order that those in the art may be better able to practice thecompositions and methods described herein, the following examples areprovided for illustration purposes.

EXAMPLES Materials and Methods

Animals

Wild type (WT) B6CBA virgin female mice and enhanced green fluorescentprotein (eGPF) transgenic male mice (C57B1/6tg(ACTbeGFP)10sb/J fromJackson Laboratories) were mated and pregnant females subjected tomid-gestation cardiac injury.). All mice used were between the ages of3-6 months. All animal care was in compliance with the Guide for theCare and Use of Laboratory Animals published by the US NationalInstitutes of Health, as well as institutional guidelines at MountSinai's School of Medicine. Initially, approximately 50 mice underwentLAD ligation surgery in order to determine the best time to induceinjury. Embryonic day (E) 12 was chosen as an earlier time point wouldcause the mother to resorb the embryos due to the hypoxic insult. If theinjury was induced later in gestation, the pregnant mouse dies due tothe hemodynamic consequences of the volume overloaded state in latepregnancy. Once it was determined that E12 was the best time to induceinjury, the survival rate was 70%. The deaths that did occur were likelydue to the infarction surgery and this survival rate matches ourpreviously published results in non-pregnant mice (Cheng et al., 2007).

DNA Extraction

Total DNA was extracted from whole maternal hearts utilizing the Bloodand Tissue DNA extraction kit (Qiagen, Valencia, Calif.).

RNA Extraction

Total RNA was extracted from cells/tissue using the Rneasy micro kit(Qiagen, Valencia, Calif.). cDNA was reverse transcribed from RNA usingthe SensiScript RT kit (Qiagen, Valencia, Calif.).

Real-time Quantitative PCR

Quantitative PCR reactions were performed with iQ™ (SYBR® GreenSupermix) on the iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules,Calif.). The PCR protocol consisted of one cycle at 95° C. (10 minutes)followed by 40 cycles of 95° C. (15 seconds) and 60° C. (1 minute). Foldchanges in gene expression were determined using the comparative CTmethod (AACt method) (Pfaffl, 2001) with normalization to ApoBendogenous control.

Primers used for RT-PCR experiments are as follow:

GFP-forward (SEQ ID NO: 1) 5″-CATCGAGCTGAAGGGCATC-3′; GFP-reverse(SEQ ID NO: 2) 5′-TGTTGTGGCGGATCTTGAAG-3′; ApoB-forward (SEQ ID NO: 3)5′-AAGGCTCATTTTCAACAATTCC-3′; ApoB-reverse (SEQ ID NO: 4)5′-GGACACAGACAGACCAGAAC-3′; Nkx2.5-forward (SEQ ID NO: 5)5′-GACAGGTACCGCTGTTGCTT-3′; Nkx2.5-reverse (SEQ ID NO: 65′-AGCCTACGGTGACCCTGAC-3′; GAPDH-forward (SEQ ID NO: 7)5″-CAGCAACAGGGTGGTGGAC-3′; and GAPDH-reverse (SEQ ID NO: 8)5′-GGATGGAAATTGTGAGGGAGATG-3′.

Comparative CT Method (ΔΔCt Method)

Briefly, the threshold cycle number (CT) was obtained as the first cycleat which a statistically significant increase in fluorescence signal wasdetected. Data was normalized by subtracting the C_(T) value of ApoBfrom that of the eGFP. Each reaction was done in triplicate and the CTvalues were averaged. The ΔΔC_(T) was calculated as the difference ofthe normalized C_(T) values (ΔC_(T)) of the treatment and controlsamples: ΔΔC_(T)=ΔC_(T treatment)−ΔC_(T control). ΔΔC_(T) was convertedto fold of change by the following formula: fold of change=2^(−ΔΔCT)(Pfaffl, 2001). The fold differences in gene expression are representedas the mean±SD. A minimum of three samples were run for each group ateach time point (n=8 for experimental group at 1 week, n=5 at 2 weeks;n=3 for shams at 1 and 2 weeks; n=4 for non-infarcted control at 1 week,n=5 at 2 weeks). The fold-differences calculated using the ΔΔC_(T)method are usually expressed as a range, which is a result ofincorporating the error of the ΔΔC_(T) value into the fold differencecalculation. The error bars represent the top and bottom range of thefold-difference. P-values were determined by a two-tailed pairedStudent's t test from the ΔC_(T) values.

Absolute Quantitation Method

Q-PCR was performed utilizing genomic DNA extracted from whole hearts. Asensitivity test (Fujiki et al., 2008; Su et al., 2008) was performed bymixing serial dilutions of DNA from GFP transgenic mouse hearts witheach of three quantities of DNA from virgin female WT mouse hearts (0,10,000, and 100,000 pg) and real-time PCR for amplification of GFP wasperformed. 1 GFP cell amongst 100,000 cells of WT background can bedetected. GFP is present as two copies per cell in the transgenic mousewe are utilizing (Joshi et al., 2008) (See FIG. 8 legend for detaileddescription).

Immunofluorescence

Maternal heart ventricular 4-μm-thick sections were fixed for 20 minutesand then blocked with 10% donkey serum (Jackson Immunoresearch, WestGrove, Pa.) for 1 hour (h) at room temperature (RT). Each section wasincubated with the primary antibody for 1 hr at RT, followed by asecondary antibody for an additional 1 h at RT and counterstained withDAPI. Finally the sections were incubated for 5 minutes with Sudan Black(0.7% in 70% EtOH) and cover-slipped with mounting media (DAKO,Carpinteria, Calif.). Slides were imaged using a Zeiss LSM-510 Metaconfocal microscope (Carl Zeiss, Munich Germany)

The following primary antibodies were used for staining: rabbit anti-GFP(ABCAM #AB6556, Cambridge, Mass.), mouse anti-alpha sarcomeric actin(Sigma #A2172, St. Louis, Mo.), mouse anti-alpha sarcomeric actinin(Santa Cruz #15335, Santa Cruz, Calif.), mouse anti-cardiac troponin-T(ABCAM #AB45932), mouse anti-alpha-smooth muscle actin (Sigma #A2547),mouse anti-smooth muscle myosin IgG (Biomedical Technologies Inc #BT562,Stoughton, Mass.), rat anti-CD31 (BD #553370, San Jose, Calif.), ratanti-VE-Cadherin (RDI #RDI-MCD144-11D4, Acton, Mass.). Alexa-488 andAlexa-568 secondary antibodies were purchased from Molecular Probes(Invitrogen, Carlsbad, Calif.).

Isolation of Maternal Cardiac Cells

Chest wall was opened to expose heart which was perfused with 10 mL PBS,using a 23-gauge needle. Entire heart was dissected out (atria andventricle) and extraneous tissue removed. Small amounts of serum-freemedium (DMEM, Cellgro, Manassas, Va.) was added to prevent heart fromdrying out. Hearts from 3-4 adult mice were minced and placed inserum-free medium. Tissue was digested with Pronase at 1 mg/ml(Calbiochem, Gibbstown, N.J.) in a spinning incubator for 1 h at 37′C.Supernatant was removed and 5 mL of warm (37° C.) complete medium (DMEMsupplemented with 10% fetal bovine serum [Cellgro, Manassas, Va.]) wasadded to the tube. (No glycine was added to inactivate the pronase, asthe serum in the medium does this). Skeletal/cardiac muscle wastriturated in the medium. During trituration, small aliquots oftendon-free solution were transferred to an empty 50 mL tube. Aboveprocedure was repeated by adding 5 mL aliquots of medium to the tubeevery few triturations until a final tendon-free volume of 35-45 mL wasachieved. Solution was filtered through a 70 micron mesh filter toremove small pieces of tendon. Filtered solution was spun at 3,000 rpmfor 5 minutes. Pellet was resuspended in 3 mL of medium then 21 mL ofred blood cell (RBC) lysis buffer (Ebiosciences, San Diego, Calif.) wasadded. After inverting a few times, filtered solution was spun at 3,000rpm for 5 min. Supernatant was removed and the pellet was resuspended in1 mL 1×PBS with antibiotics. Cells were counted.

FACS

Cells were sorted utilizing a MoFlo high speed cell sorter (DakoCytomation, Carpinteria Calif.). Both eGFP+(cells of fetal origin) andeGFP— (cells of maternal origin) populations were collected. Dataanalysis was performed using FlowJo Software (Tree Star, Ashland,Oreg.).

Flow Cytometry Cell Analysis

Analysis of specific cell markers on previously sorted eGFP+ cells wasperformed utilizing the BD LSR II (BD Biosciences, San Jose, Calif.).For intracellular cell markers cells were permeabilized using Triton-Xprior to antibody staining.

The following antibodies were used for staining: anti-Sca1 (ebiosciences#17-5981-81), anti-c-kit (Ebiosciences #27-1171-81), anti-oct4(Ebiosciences #12-5841-80), anti-nanog (Ebiosciences #51-5761-80),ant-sox2 (Millipore #MAB4343, Billerica, Mass.), Islet1 (Hybridoma bank#39.4D5-s, Iowa City, Iowa), anti-nkx2.5 (Santa Cruz #sc-14033),anti-CD31 (Santa Cruz #sc-1506), anti-CD34 (ebiosciences #56-0341-82),anti-cdx2 (Santa Cruz #19478).

Cell Culture

Differentiation of eGFP+ Cells into Endothelial Cells and Smooth MuscleCells

CMFs were prepared by isolating cardiac cells from 1 day old WT neonatalpups. Cells were enriched for CMFs by spinning at low speeds (800 rpm).The supernatant (which primarily contains CMFs) was plated for 1 hour onculture dishes to allow CMFs to attach. The supernatant, now containingresidual cardiomyocytes, was discarded. CMFs were incubated at 37° C.until confluent. CMFs were treated with Mitomycin C (MP Biomedicals.Solon, Ohio) to inhibit proliferation, incubated at 37° C. in completemedium for 24 hours and then used as feeders. FACS sorted eGFP+ cellswere cultured on the CMFs and monitored for a period of 3-4 weeks. Livecell imaging was performed using an Olympus IX-70 Live cell imagingsystem (Olympus, Center Valley Pa.).

Differentiation of eGFP+ Cells into Cardiomyocytes

Cardiomyocyte feeders were prepared by isolating cardiac cells from 1day old cyclin A2 transgenic mice as these cardiomyocytes can bepassaged and remain viable in culture indefinitely. Cells were enrichedfor cardiomyocytes by spinning at low speeds (800 rpm).

The pellet (which primarily contains cardiomyocytes) was resuspended incomplete medium and plated on culture dishes to allow residual CMFs toattach. The supernatant containing the cardiomyocytes was transferred toa new culture dish and then incubated at 37° C. Feeders were ready forexperiments after 24 hours. EGFP+ cells were cultured on cardiomyocytefeeders and monitored over a 4-5 week period. Live cell imaging wasperformed using an Olympus IX-70 Live cell imaging system (Olympus,Center Valley Pa.).

Immunofluorescence

Cells were cultured in chamber slides for 4-5 weeks and fixed with 4%paraformaldehyde (PFA) for 20 minutes and then stained. Cells wereincubated with primary antibody for 1 hour at RT, washed three times andthen incubated with a secondary antibody for an additional hour at roomtemperature. After staining, the cells were washed three times,cover-slipped with Dako mounting media and fluorescence was visualizedusing a Zeiss Axiophot2 fluorescence microscope (Carl Zeiss, MunichGermany)

Clonal Assay

Single eGFP+ cells isolated from injured maternal hearts were seeded in96-well plates containing feeders (cardiomyocytes or CMFs) with completemedium. The FACS Aria BCL2 (BD Biosciences, San Jose, Calif.) wasutilized to sort single eGFP+ cells into 96 well plates. Cells weremonitored daily to assay clonal expansion. Medium was changed every 3days. After 14 days in culture, cells were fixed using 4% PFA andsubjected to analysis.

Spectral Profile

Spectral scanning was performed using a Leica Microsystems (Leica,Mannheim, Germany) TCS SP5 confocal microscope. Images were collectedusing the lambda scan mode from 545 nm-705 nm with a 10 nm bandwidth perimage. The 543 nm HeNe laser was used for excitation and images werecollected at 512×512 pixels using the 63×/1.4NA HCX PL APO oil lens.Regions of interest (ROIs) were selected around both sample and controlcells. The mean intensity vs. wavelength for each respective ROI wasthen plotted on a graph and compared to the Alexa Fluor 568 spectralprofile.

XY Chromosome Analysis

To prepare the slide containing interphase nuclei for FISH analysis, itwas first rinsed in 2×SSC/0.1% NP-40 for 2 min at room temperature. Theslide was then dehydrated in an ethanol series and air-dried. Ready toUse (RTU) whole chromosome paint (WCP) mouse DNA probes for chromosomesX and Y (Cambio Ltd., Cambridge, UK) were mixed together and added tothe slide. The interphase nuclei and probe were co-denatured for 5minutes at 73° C. and hybridized for 48 hours at 37° C. The slide wasthen washed, to remove non-bound probe, in 0.4×SSC/0.3% NP-40 for 2 minat 72° C. and 2×SSC/0.1% NP-40 for 2 min at room temperature andair-dried. The slide was mounted with a coverslip using DAPIII/Anti-fade (Abbott Molecular, Des Plaines, Ill.). Images were obtainedusing Zeiss Axioplan 2 fluorescent microscope with CytoVision software(Genetix Corp, San Jose, Calif.). Cy3-Orange, Absorption Max→550 nm,Fluorescence Max→570 nm, FITC-Green, Absorption Max->494 nm,Fluorescence Max→520 nm.

TaqMan® Array for Pluripotent Genes

TaqMan® Array Gene Signature plates (Applied Biosystems, Carlsbad,Calif.) contain 92 assays to stem cell associated genes. Total RNA wasextracted from FACS isolated eGFP+ cells from placenta. Relative geneexpression was determined using a two-step quantitative real-time PCRaccording to the manufacturer's instructions.

Data Analysis

Statistical analysis was performed with the student's t-test.

Example 1 Fetal Cells Home to and Engraft in Injured Maternal Myocardium

Wild-type (WT) virgin female mice, age 3-6 months, were crossed withheterozygous eGFP transgenic male mice. The female mice underwentligation of the left anterior descending (LAD) artery in order to inducean anterolateral myocardial infarction (MI) at gestation day 12 (FIG.1A). This results in approximately 50% left ventricular infarction. Inaccordance with Mendelian autosomal inheritance, approximately 50% ofembryos were eGFP+.

Initially, we quantified eGFP expression in injured maternal heartsrelative to sham-operated pregnant mice and controls in which no injurywas induced. Post-partum females were sacrificed at 1 or 2 weekspost-MI. Total DNA was extracted from each total heart and eGFPexpression analyzed according to the methods described by Pfaffl, 2001(FIG. 1B). Experimental infarcted hearts harvested at 1 week post-MIcontained 120 times more eGFP than controls (p=0.0003) and 20 times moreeGFP than shams (p=0.0027; FIG. 1C). Experimental infarcted heartsharvested at 2 weeks post-MI contained 12 times more eGFP than controls(p=0.0001) and 8 times more eGFP than shams (p=0.0001) (FIG. 1D). Theabsolute numbers of eGFP cells in control, sham-operated, and MI heartswere also computed based on qPCR (FIG. 8) and 1.7% of the total heart at2 weeks post injury was composed of eGFP cells.

Example 2 Fetal Cells Adopt Diverse Cardiac Lineages In Vivo

In a separate group of infarcted and control mice, immunofluorescenceanalysis with confocal microscopy was utilized to detect eGFP+ cells inventricular tissue sections of maternal hearts at various time pointssubsequent to myocardial injury (FIG. 1B and data not shown). EGFP+cells were noted in infarct zones and peri-infarct zones of infarctedmaternal hearts at 1, 2, 3, or 4 weeks post-MI (data not shown and Table1A). Negligible numbers of eGFP cells were noted in non-infarct zones ofthe infarcted maternal hearts (Table 1B).

We further sought to determine whether the eGFP+ cells weredifferentiating into more mature cardiac cells as we noted a decrease innuclear to cytoplasmic ratio with an increase in post-injury time (datanot shown). Briefly, ventricular sections from maternal hearts analyzedat 1, 2, 3, and 4 weeks post-injury illustrated eGFP+ cells engraftingwithin infarct and peri-infarct zones. Fetal cells were positive foreGFP, nuclei were stained with DAPI, and light green backgroundfluorescence was noted in maternal cardiomyocytes.

At 3 and 4 weeks post-MI, eGFP+ cells observed in the infarct zones ofmaternal hearts also expressed markers of cardiomyocytes (α-sarcomericactin and α-actinin), smooth muscle cells α-smooth muscle actin), andendothelial cells (CD31 and VE-cadherin) (data not shown). At 3 weekspost-MI, 50% of all eGFP-positive nuclei belonged to cells that alsostained positive for -actinin, implying that 50% of eGFP cells homing tothe heart may have differentiated to cardiomyocytes (Table 1C). Theseresults suggest that fetal cells differentiated into diverse lineageswithin maternal cardiac tissue.

Table 1: cell quantification in ventricular tissue sections obtainedfrom WT female mice mated with GFP transgenic mice, subjected to cardiacinjury at mid-gestation, then sacrificed 3 weeks post-injury. 10different sections in infarct zones and 10 different sections innon-infarct zones that comprised an area of 25 sq. mm each were utilizedfor this analysis. All nuclei (detected by DAPI staining) were countedin each section. All eGFP+ nuclei were also counted and the ratios arepresented in Table 1A. This was repeated in non-infarct zones and theratios are presented in Table 1B. Alpha-actinin stained cells werecounted in the infarct zones (mononuclear) and the ratio of eGFP+ nucleithat were present in alpha-actinin stained cells is presented in Table1C.

Total nuclei A infarct zone eGFP+ nuclei % eGFP+/total nuclei 1 64 6 9.32 81 3 3.7 3 112 3 2.7 4 123 2 1.6 5 81 3 3.7 6 105 4 3.8 7 127 1 0.8 871 1 1.4 9 85 1 1.2 10 79 2 2.5 Total nuclei Non-infarct eGFP+ nucleiNon- % eGFP+ nuclei Non- B zone Infarct Zone Infarct Zone 1 72 1 1.4 293 0 0 3 84 0 0 4 101 0 0 5 147 0 0 6 62 1 1.6 7 55 0 0 8 80 0 0 9 81 00 10 64 0 0 Avg 83.9   0.2 0.2 Total eGFP+ % eGFP+ C eGFP+ nucleiActinin+ nuclei Actinin+/eGFP nuclei 1 6 4 66.7 2 3 1 33.3 3 3 2 66.7 42 1 50 5 3 1 33.3 6 4 2 50 7 1 0 0 8 1 0 0 9 1 0 0 10 2 2 100 Avg 2.61.3 50

Spectral profiles were obtained from paraffin embedded ventriculartissue sections of infarcted maternal hearts. This measure was taken, inaddition to the use of Sudan Black, to ensure that nativeautofluorescence of cardiomyocytes was not affecting fluorescence images(data not shown). In vivo analysis demonstrated that fetal cells (eGFP+)differentiated into cardiomyocytes expressing α-sarcomeric actin(α-sarc) and α-actinin, smooth muscle cells expressed α-smooth muscleactin (α-SMA) and endothelial cells expressed CD31 and VE-Cadherin(VE-cad). Paraffin embedded ventricular sections were obtained frominfarcted hearts of pregnant mice 1 week after injury; stained withrabbit anti-GFP primary antibody and donkey anti-rabbit Alexa Fluor 568secondary antibody (data not shown). Regions that represent regions ofinterest (ROIs) 1-6 were circled and subjected to spectral scanning. Themean intensities of the spectral scans for this section are plottedversus wavelength in FIG. 2. The mean intensities of the sample regionsare significantly higher than the mean intensities of the controlregions.

Example 3 Fetal Cells Isolated from Injured Maternal HeartsDifferentiate to Endothelial Cells, Smooth Muscle Cells, andSpontaneously Beating Cardiomyocytes In Vitro

We next used fluorescence activated cell sorting (FACS) to isolate fetaleGFP+ cells that had homed to maternal hearts and analyzed their invitro behavior. When plated on CMFs, we noted clonal expansion of thefetal cells, their differentiation into smooth muscle cells andendothelial cells, and the formation of vascular structures (data notshown). Other cellular phenotypes, some of which have the appearance ofneuronal cells, were also observed in these in vitro experiments withCMFs (data not shown).

Briefly, in vitro analysis of fetal cells isolated from maternal heartsdemonstrated clonal expansion on CMFs. 14 days after plating, vasculartube formation was noted in a 3-dimensional collagen matrix. Fetal cellsisolated from maternal hearts and plated on CMFs underwentdifferentiation into smooth muscle cells (α-SMA) and endothelial cells(CD31). Vascular tube formation was noted from fetal cells isolated frommaternal hearts and plated on CMFs with expression of α-SMA and CD31.Fetal cells isolated from maternal hearts and plated on Cyclin A2neonatal cardiomyocytes differentiated into beating cardiomyocytes.Cardiomyocytes arising from fetal cells isolated from maternal heartsexpressed cardiac troponin T (cTnT) and connexin 43 (Cx43).

As we did not observe differentiation of fetal cells into cardiomyocyteson CMFs, we utilized cardiomyocytes isolated from neonatal cyclin A2transgenic mice (Cheng et al., 2007) as feeders. When plated on thesefeeders with standard medium consisting of DMEM supplemented with FBS,the isolated eGFP+ fetal cells differentiated into spontaneously beatingcardiomyocytes (about 48 beats/minute, data not shown). The resultinglineages also expressed cardiac troponin T (data not shown). Furtheranalysis of eGFP+ fetal cells cultured for 5 weeks in chamber slidesindicated expression of the gap junction marker connexin 43 (data notshown). This provides compelling evidence for formation ofelectromechanical connections between the cardiomyocytes derived fromeGFP+ fetal cells and the feeder cardiomyocytes.

Example 4 Fetal Cells Exhibit Clonality and Undergo CardiacDifferentiation in a Fusion-Independent Manner

Clonal analysis was performed to confirm the ‘sternness’ of the fetalcells giving rise to cardiac cells (FIG. 3A). FACS for eGFP+ cells wasperformed and single cells were seeded in 96-well plates containing WTneonatal cardiomyocytes as feeders. Clones derived from eGFP+ fetalcells were expanded for 14 days and total clones counted in each colony.Two 96-well plates were utilized and 4 wells in each plate gave rise tocolonies after 7 days (approximately 50% of the wells in each platecontained viable cells at this time point) yielding an approximatecloning efficiency of 8.3%. The number of cells identified on days 6, 7,9, 12, 13 and 14 is provided in FIG. 3B.

In order to mechanistically assess whether fusion, rather thandifferentiation, was the cause of the appearance of eGFP+ cardiomyocytesin our in vitro assays, we analyzed the number of nuclei present withinour fetal cell-derived cardiomyocytes and consistently noted that thesecardiomyocytes were mononuclear (data now shown). Where only GFP andDAPI staining was seen, still pictures were taken depicting the beatingof these cells (data not shown). Cardiomyocytes derived in vitro fromfetal cells isolated from maternal heart were found to be mononuclear.

Furthermore, fluorescence in situ hybridization (FISH) for X- andY-chromosomes revealed one set of sex chromosomes within the eGFP+cardiomyocyte nuclei, establishing the diploid nature of these nucleiand effectively ruling out fusion between eGFP+ fetal cells and feedercardiomyocytes as the source of eGFP+ cardiomyocytes (data not shown).Briefly, fetal cell-derived cardiomyocytes had diploid nuclei with oneset of sex chromosomes detected per cell. eGFP+ cells (488 nm) thatdifferentiated into cardiomyocytes as determined with cTNT staining (568nm) were observed. The same cells were observed with different red andgreen wavelength filters to detect the X chromosome (520 nm) and the Ychromosome (550 nm). A tetraploid nucleus was observed in a non-eGFPcell. Only the nuclei of the cells and as the X, Y probes exhibitedfluorescence at different wavelengths (FITC: 520 nm, Cy₃: 550 nm) andtheir signals could be easily distinguished from the green fluorescenceof the GFP (Alexa 488: 488 nm) and the secondary antibody to cardiactroponin T (Texas Red: 568 nm). The ability to detect tetraploid nucleiwith this assay was demonstrated by identifying cells that were found ina region where GFP cells were not detected.

Example 5 Fetal Cells Selectively Home to the Injured Maternal Heart andnot to Non-Injured Organs

To assess whether eGFP+ cells from the fetus were homing selectively tothe injured heart, we utilized FACS to sort eGFP+ cells from a varietyof organs and tissues harvested from pregnant mice subjected to cardiacinjury. These organs and tissues were minced and triturated to generatecell suspensions (FIG. 4A). Corresponding cell populations were obtainedfrom age-matched pregnant WT female mice mated with WT males and used ascontrols to establish the appropriate FACS gates to select eGFP+ cells.Cells were isolated from the injured heart, blood, skeletal muscle,chest wall, eGFP-littermates, liver, lung, and placenta.

FACS to select eGFP+ cells was performed at two time points, 4.5 dayspost-injury (prior to delivery) and 7 days post-injury (after delivery)for all of these tissues except placenta (analyzed before delivery only)as it is resorbed by the mother in mice at time of delivery (FIG. 4B).The low quantity of eGFP+ cells in all tissues, including injured heart,prior to 4.5 days post injury precluded any detailed phenotypicanalyses. Therefore, it appears that mobilization of fetal cells inresponse to maternal injury takes approximately 4.5 days. In the injuredheart, ˜1.1% of the cells were eGFP+ prior to delivery and this numberrose significantly to ˜6.3% just after delivery. In blood, ˜1.3% ofcells were eGFP+ before delivery and this number rose to ˜3.6% afterdelivery, although this increase was not statistically significant.

Delivery therefore seems to cause the numbers of fetal cells enteringthe maternal circulation to rise, and this corresponds with asignificant increase in fetal cells homing to the injured heart. Therewere negligible numbers of eGFP+ cells noted in skeletal muscle beforeand after delivery. The chest wall, where a lesser degree of injury wasinduced as an incision had to be performed to induce cardiac injury,exhibited a relatively smaller percentage of fetal cells compared withheart and blood. There was no increase in the number of fetal cellshoming to the chest wall after delivery, likely due to healing in theseven days post-injury. EGFP-littermates were also examined for thepresence of eGFP+ cells. Although a few cells were noted prior todelivery likely due to the shared circulation with the eGFP+littermates, eGFP cells were not detected in these littermates afterdelivery. Liver and lung exhibited negligible numbers of fetal cells. Asexpected, placenta exhibited the largest percentage of eGFP cells withapproximately 36% of placenta cells expressing eGFP. Overall, theresults provide clear evidence for the selective and specific homing ofeGFP+ fetal cells to the injured heart of the mother, and not to othernon-injured maternal tissues (FIG. 4B and FIG. 4C).

Example 6 Fetal Cells Isolated from Maternal Hearts Express a Variety ofProgenitor Markers, Most Notably Cdx2

To establish the identity of the cell type(s) involved in fetal maternaltransfer, we analyzed FACS-sorted, eGFP+ cells isolated from maternalhearts 1 week post-injury for stem/progenitor cell markers (FIG. 5A).80% of these cells expressed Nkx2. (Komuro and Izumo, 1993; Lints etal., 1993; Ueyama et al., 2003) implying that cardiomyogenicdifferentiation had begun as soon as these cells entered the injuredmaternal heart. Consistent with this, very small numbers, <1%, ofplacental cells from pregnant mice expressed Nkx2.5 (FIG. 9).Additionally, 46% of cells homing to the maternal heart expressed CD31,which was not surprising given the degree of fetal cell-mediatedvasculogenesis we observed in injured maternal hearts. The marker Cdx2was found in 38% of fetal cells.

Cdx2 regulates trophoblast stem (TS) cell development and proliferation(Niwa et al., 2005; Strumpf et al., 2005), but the present inventorsidentified for the first time that Cdx2 is associated withcardiomyogenic differentiation. This finding raises the possibilitythat, in the setting of acute injury, TS cells from placenta can giverise to various cardiac lineages in addition to forming placenta. Fetalcells isolated from maternal hearts also displayed lower levels ofseveral markers of endogenous cardiac progenitors, namely Sca-128, 29(21%), cKit (25%) and Islet1 (3%), as well as embryonic stem (ES) cellmarkers Pou5f1 (2%), Nanog (3%) and Sox2 (24%). The higher expression ofSox2 is consistent with its expression in non-ES cells as well. Finally,hematopoietic stem cell factor CD34 was expressed in 15% of the eGFP+cells which is consistent with placenta acting as a rich source ofhematopoietic stem cells (FIG. 5A).

As the eGFP+ cells were traversing through or derived from the placenta,we analyzed gene expression of known ‘sternness’ factors in eGFP+ cells.We sorted eGFP+ cells from end-gestation placentas from three differentpregnant mice that had been subjected to myocardial injury. RNAexpression of 92 known pluripotency genes was analyzed (Table 2), andgene expression relative to GAPDH expression for the most prevalenttranscripts is plotted in FIG. 5B. These mRNA array studies confirmedthe presence of Cdx2 and Eomesodermin (Eomes), another marker of TScells, in the eGFP+ placenta cells.

Example 7 Discussion

The selective homing of eGFP+ cells in our model to the site of maternalcardiac injury with lack of such homing to non-injured tissues points tothe presence of precise signals sensed by cells of fetal origin thattarget them to diseased myocardium specifically, and to differentiateinto diverse cardiac lineages (FIG. 6). Most notable is theirdifferentiation into functional cardiomyocytes that are able to beat insyncytium with neighboring cardiomyocytes (data not shown), thus a fetusmay assist in protecting the mother's heart during and after pregnancy.The present studies were inspired by the recovery noted in peripartumcardiomyopathy, whereby a remarkable 50% of women spontaneously recoverfrom heart failure. Peripartum cardiomyopathy has the highest rate ofrecovery amongst all etiologies of heart failureIt was this observationthat prompted us to hypothesize that there may be a fetal or placentalcontribution to counteract maternal cardiac injury. The mouse injurymodel presented herein serves as a model system of murine fetomaternalmicrochimerism that can help identify appropriate cell types for cardiacregeneration.

To this end, a far greater spectrum of potential applications to thefield of heart disease emerges from the present studies. The challengeof cardiovascular regenerative medicine is to develop novel therapeuticstrategies to facilitate regeneration of normally functioningcardiomyocytes in the diseased heart. Utilizing live imaging, we havedemonstrated that fetal cells differentiate into spontaneously beatingcardiomyocytes after homing to the heart.

Our identification of Cdx2 as a unique and highly prevalent markerexpressed on fetal cells in the maternal myocardium offers a newperspective regarding the appropriate cell type that may be used fortherapeutic purposes. The Cdx family of transcription factors consist ofthree mouse homologues (Cdx 1, 2, and 4) of the Drosophila caudalhomeobox genes, which are involved in specifying cell position along theanteroposterior axis, with similar functions in the later developmentalstages of the mouse embryoas well as morphological specification ofmurine gut endoderm. Cdx2 is also involved in trophectoderm fatecommitment in the developing blastocyst. The trophectoderm gives rise tothe trophoblast stem cells which have previously been associated solelywith differentiation to the placenta lineage.

Our results point towards the transfer of several populations ofprogenitor cells, and our finding of Cdx2 cells of fetal or placentalorigin in the heart indicates a new method of treating cardiacconditions with a Cdx2 cell type that is capable of cardiacdifferentiation under injury conditions that can be readily isolatedfrom placenta.

TABLE 2 Raw Ct Values Values Normalized to Gene ID Placenta1 Placenta2Placenta3 Placenta1 Placenta2 Placenta3 18S-Hs99999901_s1 6.3705 8.4705316.0094 Gapdh- 20.9696 18.8751 13.6232 0 0 0 Mm99999915_g1 Hprt1-25.9639 23.6467 33.147 4.9943 4.7716 19.5238 Mm00446968_m1 Gusb- 26.459524.424 33.4364 5.4899 5.5489 19.8132 Mm00446953_m1 Actc1- 28.888125.4953 22.127 7.9185 6.6202 8.5038 Mm01333821_m1 Afp-Mm00431715_m120.0476 18.6008 12.1797 −0.922 −0.2743 −1.4435 Bxdc2- 27.1527 24.370920.021 6.1831 5.4958 6.3978 Mm00503229_m1 Cd34- 26.2739 23.7997 18.42775.3043 4.9246 4.8045 Mm00519283_m1 Cd9-Mm00514275_g1 24.2361 21.335217.3176 3.2665 2.4601 3.6944 Cdh5- 23.3841 20.926 15.4999 2.4145 2.05091.8767 Mm00486938_m1 Cdx2- 27.8661 27.2999 21.1473 6.8965 8.4248 7.5241Mm00432449_m1 Col1a1- 20.7192 18.2277 13.2472 −0.2504 −0.6474 −0.376Mm00801666_g1 Col2a1- 26.1934 27.0833 19.0532 5.2238 8.2082 5.43Mm00491889_m1 Commd3- 30.2898 27.3424 24.3104 9.3202 8.4673 10.6872Mm00521684_m1 Crabp2- 29.5281 27.8679 22.113 8.5585 8.9928 8.4898Mm00801693_g1 Ddx4- 33.419 31.4264 27.0787 12.4494 12.5513 13.4555Mm00802445_m1 Des-Mm00802455_m1 27.1446 26.0115 20.7865 6.175 7.13647.1633 Dnmt3b- 29.001 27.4062 22.5412 8.0314 8.5311 8.918 Mm01240113_m1Lefty1- UD 36.3593 31.8521 UD 17.4842 18.2289 Mm00438615_m1 Eomes-34.1758 32.7162 27.0058 13.2062 13.8411 13.3826 Mm01351985_m1 Fgf4- UDUD UD UD UD UD Mm00438917_m1 Fgf5- 35.3186 36 30.7142 14.349 17.124917.091 Mm00438919_m1 Flt1-Mm00438980_m1 26.067 24.1073 19.1198 5.09745.2322 5.4966 Fn1-Mm01256744_m1 21.7467 19.7424 14.3723 0.7771 0.86730.7491 Foxa2- 28.0793 25.4683 25.0441 7.1097 6.5932 11.4209Mm01976556_s1 Foxd3- UD 30.8118 UD UD 11.9367 UD Mm02384867_s1 Gabrb3-35.8833 32.5907 28.297 14.9137 13.7156 14.6738 Mm00433473_m1Gal-Mm00439056_m1 33.0773 29.2917 24.8623 12.1077 10.4166 11.2391 Gata4-26.0794 25.4503 19.0996 5.1098 6.5752 5.4764 Mm00484689_m1 Gata6- 26.98824.3801 19.2929 6.0184 5.505 5.6697 Mm00802636_m1 Gbx2- UD 31.481627.8815 UD 12.6065 14.2583 Mm00494578_m1 Gcg-Mm00801712_m1 UD 38.193733.0883 UD 19.3186 19.4651 Gcm1- 31.5349 31.052 23.4502 10.5653 12.17699.827 Mm00492310_m1 Gdf3- 36.7057 34.4247 29.3908 15.7361 15.549615.7676 Mm00433563_m1 Gfap- 36.3519 35.458 28.0648 15.3823 16.582914.4416 Mm00546086_m1 Grb7- 28.2935 26.5652 21.7775 7.3239 7.6901 8.1543Mm01306734_m1 Hbb-b2- 20.7765 20.2892 13.2353 −0.1931 1.4141 −0.3879Mm00731743_mH Hba-x- 23.6843 25.7335 16.204 2.7147 6.8584 2.5808Mm00439255_m1 Mnx1- 37.833 33.6542 29.1002 16.8634 14.7791 15.477Mm00658300_g1 Iapp-Mm00439403_m1 UD UD 34.065 UD UD 20.4418 Ifitm1-26.5783 23.0079 21.176 5.6087 4.1328 7.5528 Mm00850040_g1 Ifitm2-27.9784 24.3113 24.372 7.0088 5.4362 10.7488 Mm00850080_g1I16st-Mm00439668_m1 26.2689 24.1603 19.5894 5.2993 5.2852 5.9662 Igfbp2-26.9526 25.3066 20.1445 5.983 6.4315 6.5213 Mm00492632_m1Ins2-Mm00731595_gH UD 30.2597 28.4544 UD 11.3846 14.8312 Pdx1- UD35.5069 31.2635 UD 16.6318 17.6403 Mm00435565_m1 Isl1-Mm00627860_m138.4946 33.7686 28.5575 17.525 14.8935 14.9343 Kit-Mm00445212_m1 26.212125.2267 19.107 5.2425 6.3516 5.4838 Krt1-Mm00492992_g1 30.9432 33.895424.0382 9.9736 15.0203 10.415 Lama1- 23.3831 23.2048 15.6915 2.41354.3297 2.0683 Mm00439445_m1 Lamb1-1- 24.1153 21.8563 16.8898 3.14572.9812 3.2666 Mm00801853_m1 Lamc1- 23.7416 21.9146 16.7137 2.772 3.03953.0905 Mm00711820_m1 Lefty2- UD 37.5112 30.8274 UD 18.6361 17.2042Mm00774547_m1 Lifr-Mm00442940_m1 24.8734 23.0875 17.5635 3.9038 4.21243.9403 Lin28- 30.6329 30.7133 23.7301 9.6633 11.8382 10.1069Mm00524077_m1 Myf5- UD UD UD UD UD UD Mm00435125_m1 Myod1- 36.721433.2402 33.0302 15.7518 14.3651 19.407 Mm00440387_m1 Nanog- 37.034426.5706 27.9819 16.0648 7.6955 14.3587 Mm02019550_s1 Nes-Mm00450205_m128.063 27.425 21.2942 7.0934 8.5499 7.671 Neurod1- UD 28.183 30.9275 UD9.3079 17.3043 Mm01946604_s1 Nodal- 33.4446 32.016 26.1958 12.47513.1409 12.5726 Mm00443040_m1 Nog-Mm00476456_s1 27.4575 25.9114 20.74416.4879 7.0363 7.1209 Nppa- 35.4623 33.6554 29.1081 14.4927 14.780315.4849 Mm01255747_g1 Nr5a2- 34.1867 33.3681 27.3547 13.2171 14.49313.7315 Mm00446088_m1 Nr6a1- 30.1965 28.0504 23.2667 9.2269 9.17539.6435 Mm00599848_m1 Olig2- 36.607 37.1301 29.3012 15.6374 18.255 15.678Mm01210556_m1 Pax4- UD UD UD UD UD UD Mm01159036_m1 Pax6- 36.557634.7341 30.4868 15.588 15.859 16.8636 Mm00443072_m1 Pecam1- 24.494422.4209 17.2251 3.5248 3.5458 3.6019 Mm00476702_m1 Podxl- 24.790424.0435 17.0374 3.8208 5.1684 3.4142 Mm00449829_m1 Pou5f1- 35.266232.652 29.28 14.2966 13.7769 15.6568 Mm00658129_gH Pten-Mm00477210_m127.204 24.7365 20.3668 6.2344 5.8614 6.7436 Ptf1a- UD UD UD UD UD UDMm00479622_m1 Rest-Mm00803268_m1 27.8035 25.3074 20.7451 6.8339 6.43237.1219 Runx2- UD 36.1486 31.7123 UD 17.2735 18.0891 Mm00501578_m1Sema3a- 32.1638 29.0252 25.3798 11.1942 10.1501 11.7566 Mm00436469_m1Serpina1a- 31.0904 26.3099 25.5911 10.1208 7.4348 11.9679 Mm02748447_g1Sfrp2- 29.387 26.3426 22.2145 8.4174 7.4675 8.5913 Mm00485986_m1 Sox17-27.3797 27.152 20.0432 6.4101 8.2769 6.42 Mm00488363_m1Sox2-Mm00488369_s1 UD 27.3828 26.4852 UD 8.5077 12.862 Sycp3- 36.767332.6623 29.1498 15.7977 13.7872 15.5266 Mm00488519_m1 Syp-Mm00436850_m133.0295 30.0902 24.2542 12.0599 11.2151 10.631 T-Mm00436877_m1 UD UD39.9782 UD UD 26.355 Tat-Mm01244282_m1 35.7324 36.7088 30.4848 14.762817.8337 16.8616 Tdgf1- UD 27.1602 28.2507 UD 8.2851 14.6275Mm00783944_g1 Tert-Mm00436931_m1 32.5026 30.957 26.0882 11.533 12.081912.465 Tcfcp2l1- 31.0598 30.9915 24.7141 10.0902 12.1164 11.0909Mm00470119_m1 Th-Mm00447546_m1 UD 37.1873 30.4919 UD 18.3122 16.8687Utf1-Mm00447703_g1 31.1389 28.538 25.2141 10.1693 9.6629 11.5909Wt1-Mm00460570_m1 31.4399 30.056 24.2453 10.4703 11.1809 10.6221Xist-Mm01232884_m1 27.0304 24.1311 19.2598 6.0608 5.256 5.6366 Zfp42-29.3088 25.9252 23.1699 8.3392 7.0501 9.5467 Mm01194090_g1Eras-Mm01345955_s1 UD 28.1246 28.6338 UD 9.2495 15.0106 Raf1- 27.213125.4176 19.9228 6.2435 6.5425 6.2996 Mm00466513_m1 Ctnnb1- 23.505621.6847 15.913 2.536 2.8096 2.2898 Mm00483033_m1 Eef1a1- 19.9088 16.64813.2675 −1.0608 −2.2271 −0.3557 Mm01966109_u1 UD = Undetermined

The following example describes isolation of Cdx2 cells fromend-gestation mouse and human placentas utilizing lentiviral vectors andtesting their differentiation properties in vitro. A lentivirus wasconstructed in which the murine Cdx2 promoter drives expression of thereporter gene tdTomato. The control lentivirus employs a cytomegaloviruspromoter driving tdTomato. Cell suspensions of placenta tissues are madeand Cdx2 cells are sorted based on the red fluorescence of tdTomato.Single cell sorting into 96-well plates is performed to confirmclonality. These are then be cultured on cardiac mesenchymal fibroblastsand neonatal cardiomyocytes to test their ability to differentiate intoendothelial cells, smooth muscle cells, and cardiomyocytes. Live-imagingmicroscopy is utilized to assess spontaneous beating of Cdx2cell-derived cardiomyocytes.

The following example describes testing the ability of Cdx2 cellsisolated from placenta to form cardiomyocytes and blood vessels in vivovia transplantation experiments in the post-myocardial infarctionsetting. We will first test Cdx2 cells' cardiovascular differentiationpotential in vivo and their ability to restore cardiac function in arodent model Immunohistochemical approaches are utilized to detectformation of endothelial cells, smooth muscle cells, and cardiomyocytesin infarcted hearts. Cardiac function enhancement is detected withmagnetic resonance imaging (MRI). If transplantation of Cdx2 cells intoinfarcted rodent hearts demonstrates evidence of cardiac regenerationwith improvement in ejection fraction, a large animal study is to beperformed in an art-recognized porcine infarct model. Porcine (pig)infarct models include those described by, for example, Hayase et al.(2005) Heart Cir. Physiol. 288: H2995-H3000. Briefly, hearts of livingpigs are treated a cell population containing Cdx2 cells by injectioninto one or more sites surrounding an infarction. Alternatively, heartsof living pigs are injected with a vector (e.g., a lentiviral vector oradenoviral vector) encoding Cdx2 by injection into one or more sitessurrounding an infarction.

The following example describes isolation of Cdx2 cells from humanplacenta tissues and determination of new cell surface markers usefulfor sorting of these cells for further translational studies. Cells maybe sorted based on unique cell surface markers instead of reportergenes. Cdx2 cells may be isolated from human placenta tissues andproteomic approaches employed to identify cell surface markers that maybe utilized for FACS sorting. Membrane fractionation of Cdx2 cellssorted using the lentiviruses constructed above may be carried outfollowed by mass spectrometry to identify new peptides. Antibodies tothese peptides are designed and tested for their ability to identify andsort Cdx2 cells.

Example 8 Isolation of Cdx2 Cells and a Heterogeneous Mix ofFetal-Derived Placenta Cells from End-Gestation Mouse and HumanPlacentas, Testing of their Differentiation Properties In Vitro, andIdentification of New Cell Surface Markers that Facilitate FurtherSorting

A lentivirus has been constructed in which the murine Cdx2 promoterdrives the expression of tdTomato, and the corresponding controllentivirus utilizes the CMV promoter to drive expression of tdTomato(FIG. 10A, 10B). As there is no definitive characterization of knowncell surface markers distinctly associated with the transcription factorCdx2 in placenta stem cells, FACS sorting is conducted utilizing alentivirus driving a fluorescent reporter.

In prior studies, permeabilized GFP cells were isolated from maternalhearts in order to assay the presence of Cdx2 by FACS. The efficacy ofour lentivirus in its ability to select Cdx2 cells has been demonstratedin FIG. 10C. When the CT26. A wild type (WT) murine colon carcinoma cellline which expresses Cdx2 was transduced with the lentivirus; 40% ofcells expressed TdTomato. This is more efficient than the 17% of cellsidentified by the Cdx2 monoclonal antibody through FACS sorting depictedin FIG. 10D.

The expression of Cdx2 and Eomesodermin, two markers of TS cells, havebeen confirmed by RNA microarray in fetal cells isolated fromend-gestation placentas (see FIG. 5C).

Cdx2 cells may be specifically selected from end-gestation placentasfrom both mouse and human species and their differentiation propertiesin vitro may be compared to a heterogeneous mix of fetal-derivedplacenta stem cells (hfpcs). Interestingly, the RNA array data indicatesthat fetal cells in the placenta express high levels of CD9 (FIG. 5C), acell surface molecule and a member of the transmembrane-4 family thathas been associated with Cdx2 expression in intestinal epithelial cells.Membrane fractionation of Cdx2 cells from placenta sorted using thelentiviruses described above is performed, using an antibody to CD9 toconfirm its expression on the surface of these cells, and utilizing massspectrometry to identify other novel peptides, if needed.

End-gestation placenta from both mice and humans isminced and ahomogeneous cell suspension prepared according to established protocols.The cell suspension is mixed with lentivirus for a period of at least 24hrs and then FACS is utilized to collect cells expressing tdTomato. Thecontrol lentivirus is utilized in separate tubes of the correspondingcell suspension to set the appropriate gates for FACS. Cdx2-positivecells are then co-cultured on feeder layers of cardiac mesenchymalfibroblasts (CMF) and separately, on feeder layers of neonatalcardiomyocytes. Live-imaging microscopy is utilized to document thedifferentiation pathways of the Cdx2 cells. Immunofluorescence stainingwith antibodies to VE-Cadherin, CD31, alpha-smooth muscle actin,alpha-actinin, and cardiac troponin T is performed to assay fordifferentiation to the endothelial cell, smooth muscle cell, orcardiomyocyte fate once the cells are fixed.

Hfpcs are sorted from cell suspensions of end-gestation mouse placentasutilizing the GFP marker by mating wild-type virgin mice withGFP-expressing male transgenic mice, thus assuring that approximately50% of fetuses express GFP. These are also co-cultured with the feederlayers described above and their differentiation characteristics aremonitored also as described above. These experiments allow fordetermination of the precise cell type(s) that is(are) capable ofdifferentiating to spontaneously beating cardiomyocytes in vitro asrecently demonstrated.

This is important to the field of regenerative biology given thetechnical difficulties of inducing true cardiomyogenesis in vitro.

Antibodies to CD9 will be used as described above to confirm itsexpression on Cdx2 cells isolated from murine and human placenta, and toexclude its expression on the cell surfaces of other stem cell typesisolated from placenta. High throughput proteomics studies, such as twodimensional gel electrophoresis, liquid chromatography, and massspectrometry, will also be used in order to identify cell surfacemarkers of Cdx2 cells if CD9 does not appear to be a unique cell surfacemarker expressed by Cdx2 cells from placenta.

Example 9 Testing the Ability of Cdx2 Cells Versus a Heterogeneous Mixof Fetal-Derived Cells Isolated from Placenta to Form Cardiomyocytes andBlood Vessels In Vivo and Restoring Cardiac Function Via TransplantationExperiments in the Post-Myocardial Infarction Setting.

a) Experimental Plan-Studies in the Rat MI Model:

Nude, athymic, female rats are utilized for these experiments with fourgroups to be tested as follows: 1) Group 1 will undergo MI via ligationof the left anterior descending artery (LAD). This results in a 30%infarction of the left ventricle as previously demonstrated (Woo et al,2006). After allowing one week of recovery, the chest will be re-openedand the animals will receive Cdx2 cells that have been collectedutilizing the Cdx2 lentivirus from end-gestation rat placentas obtainedfrom a different group of female rats. 10⁶ cells will be injected intothe peri-infarct border zone at ten distinct, equally spaced sites. Celldelivery will be performed in a blinded manner. The chest will beclosed, and the animals will be monitored over a 3-month period.Echocardiography will be utilized to assess cardiac function atbaseline, 1 week after MI prior to cell injection, 1 month post cellinjection, and at 3 months post-cell injection. The animals will thenundergo hemodynamic studies with LV pressure catheters prior tosacrifice at 3 months post cell injection. Hearts will be collected andinfarction sizes will be measured using Masson Trichrome stainingaccording to conventional methods (Woo et al, 2006). Tissue sectionswill be prepared from infarct zone, peri-infarct border zone and remotezones; and cellular differentiation will be analyzed usingimmunofluorescence. Antibodies to VE-Cadherin, CD31, alpha-smooth muscleactin, alpha-actinin, and cardiac troponin T will be utilized to assaydifferentiation to endothelial, smooth muscle and cardiac lineages.Co-immunostaining with antibody to tdTomato will be performed toascertain whether the Cdx2 cells give rise to these diverse cardiaclineages in the in vivo infarct model. All echocardiographic,hemodynamic, and histologic studies will be performed in a blindedmanner. 2) Group 2 will undergo MI via LAD ligation. One week later,they will receive 10⁶ hfpcs via direct injection in the peri-infarctborder as described above. The hfpcs will be obtained from theend-gestation placentas of a different group of female rats, andmononuclear cells will be isolated utilizing FACS sorting based on thepresence of the Y-chromosome to ensure they are of fetal origin.Echocardiographic, hemodynamic, and histologic analyses will beperformed as described above. 3) Group 3 will undergo MI via LADligation and one week later, will receive 10⁶ rat cardiac fibroblasts.This will ensure that any evidence of cardiac repair we note in Groups 1and 2 is due to the presence of stem or progenitor cells and not due tononspecific cell effects of preventing scar expansion. Echocardiography,hemodynamic, and histologic analyses will be performed as describeabove. 4) Group 4 will undergo MI via LAD ligation and one week later,will receive PBS control injections in ten (10) distinct peri-infarctsites. Echocardiography, hemodynamic, and histologic analyses will beperformed as describe above.

Power Analysis:

Power for balanced, one-way ANOVA for 4 groups (Control, FibroblastControl Cells, Mixed Placental Cells and Cdx2 Placental Cells) with N=12in each group at a fixed alpha level of 0.05 using the Omnibus Overall FTest for One-Way ANOVA. The power is 0.875 or 87.5% for the Omnibus Testthat at least one group mean is different from the others. The power todetect differences in (specific) pair-wise group means is 0.816 or81.6%.

b) Experimental Plan-Studies in the Porcine MI Model:

Porcine Infarction Model:

Forty (40) Yorkshire swine weighing 20-25 kg will undergo surgicalinstrumentation for subsequent noninvasive measurement of LV pressureand dimension and myocardial oxygen consumption according toconventional methods (Amado et al., 2005; 1 Ekelund et al., 1999; andSaavedra et al., 2002). The animals will be instrumented, via mediansternotomy, with indwelling catheters in the descending aorta, rightatrial appendage, and great cardiac vein. Endocardial ultrasoundcrystals (Sonometrics, Ontario, Canada) will be inserted to measureshort-axis dimension, and a pneumatic occluder will be placed around theinferior vena cava for graded preload reduction to assessLV-pressure—dimension relations. A 4-5 mm flow probe (Transonics,Ithaca, N.Y.) will be placed around the mid-LAD to measure coronaryvolume flow. A solid-state miniature pressure transducer (P22,Konigsberg Instruments, Pasadena, Calif.) will be placed in the LV apexfor high-fidelity recordings of LV pressure. Additional pacing leadswill be secured in the left atrial appendage for pacing duringhemodynamic measurements. During surgery, MI will be induced by a 60-minocclusion of the LAD, followed by reperfusion. There will be 12 animalsper group and three groups will be tested. Animals will be randomized toreceive intramyocardial injections of either human Cdx2 cells or humanhfpcs (depending on the best results group from rat study) [10⁶ cells].The second group of animals will receive 10⁶ human fibroblasts, and thethird will receive PBS control injections. All injections will beperformed in a blinded manner. In order to preclude the possibility ofimmune rejection for the purposes of this study, an immunosuppressiveregimen of IV prednisone and IV tacrolimus/cyclosporine will be utilizedaccording to prior experience of the SJTR1 team in a different stem celltherapy study. Four additional animals will undergo surgical preparationand instrumentation without coronary occlusion for the assessment ofhemodynamic values in the absence of MI. Hemodynamics in the infarctedanimals will be re-assessed six weeks post-MI.

Power Analysis:

Power for balanced, one-way ANOVA for 3 groups (Control Vehicle, ControlCells, Cdx2 Cells) with N=12 in each group at a fixed alpha level of0.05 using the Omnibus Overall F Test for One-Way ANOVA. The power is0.902 or 90.2% for the Omnibus Test that at least one group mean isdifferent from the others. The power to detect differences in (specific)one group mean is different from the other group mean is 0.856 or 85.6%.

Measures of Hemodynamics and Cardiac Function:

Pressure-dimension data will be recorded at steady state and duringtransient inferior vena cava occlusion. Myocardial contractility and/orwork will be indexed by the maximal rate of isovolumetric contraction(+dP/dt), stroke work (SW), and ventricular elastance, the slope of theend-systolic pressure—dimension relationship (Ees) (Hare et al., 1999).Preload will be analyzed as end-diastolic dimension and pressure, andafterload will be evaluated as effective arterial elastance (Kelly etal., 1992), the ratio of LV end-systolic pressure to stroke dimension.Hemodynamic pressure-dimension data will be digitized at 200 Hz andstored for subsequent analysis on a personal computer by using customsoftware. Myocardial oxygen consumption per cardiac cycle (MVO2) will becalculated from the arteriovenous difference of oxygen saturation insimultaneously sampled coronary sinus and aortic blood, multiplied byLAD flow and divided by heart rate. Cardiac mechanical efficiency willbe calculated as the SW/MVO2 ratio (Ekelund et al., 1999). Transthoracicechocardiography (SONOS 5500) and MRI (GE 1.5 T Cardiac MagneticResonance stand with a short 1.2 meter bore and body coils optimized forF-19) will be utilized to assess ventricular function prior to MI andsix weeks post-MI.

Immunofluorescence Studies for Detection of Progenitor Cells andCellular Differentiation:

Three (3) animals from each group will be sacrificed two weeks post-MIand the remainder will be sacrificed six-weeks post-MI. Their heartswill be analyzed at both microscopic and gross levels. Myocardial tissuewill be prepared for immunohistochemistry according to conventionalmethods (Kawamoto et al, 2004; and Shake et al., 2002). Tissue sampleswill be obtained from three specific areas: infarct zone, peri-infarctborder zone, and remote tissue. Antibodies to VE-Cadherin, CD31,alpha-smooth muscle actin, alpha-actinin, and cardiac troponin T will beutilized to assay differentiation to endothelial, smooth muscle andcardiac lineages. Antibody to tdTomato will be utilized to identify theCdx2 cells to determine if these cells engraft and differentiate todiverse cardiac lineages after transplantation. Phosphohistone H3staining will be performed to assess the presence of active dividingcells.

Infarct Size Assessment and Myofilament Density Measurement:

Myocardial fibrosis will be determined as a percentage of the leftventricle from whole-heart slices. For this purpose, hearts will beexcised and sectioned into 8-mm-thick short-axis slices. Each slice willbe weighed and digitally photographed. Analysis will be performed usingMasson trichrome staining using conventional methods (Cheng et al.,2007). Infarcted areas and LV borders will be manually traced for eachslice by using a custom research software package IMAGE ANALYSIS 4.0.2beta version (Scion, Frederick, Md.). Infarct size will be determined,in a blinded fashion, as percentage of LV mass from the digital picturesand normalized by the weight of the slice. Myofilament density will bemeasured at the border zones by counting the numbers of cardiomyocytes(delineated by immunostaining for a-actinin) per high-power field andaveraging over at least three mid-ventricular transverse sections perheart (Woo et al., 2006).

Statistical Analyses:

All values will be expressed as mean±SE. Student's paired t test will beused to compare data before and after treatment. A value of p<0.5 willbe considered as statistically significant.

While preferred embodiments have been shown and described herein, itwill be obvious to those skilled in the art that such embodiments areprovided by way of example only. Numerous variations, changes, andsubstitutions will now occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed in practicing the invention. It is intended that the followingclaims define the scope of the invention and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

Abbreviations and Acronyms:

eGFP enhanced green fluorescent protein; TS trophoblast stem; Cdx2Caudal-related homeobox2; ES embryonic stem; RT room temperature; WTwild type; MI myocardial infarction; FACS fluorescence activated cellsorting; CMFs cardiac mesenchymal feeders; VE-cad VE-Cadherin; ROIsregions of interest; α-sarc alpha-sarcomeric actin; cTnT cardiactroponin T; Cx43 connexin 43; and α-SMA alpha-smooth muscle actin.

REFERENCES CITED

All publications, patents, patent applications, and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentinvention.

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1. A composition comprising a population of cells and a pharmaceuticallyacceptable carrier for increasing cardiomyocyte formation, increasecardiomyocyte proliferation, increase cardiomyocyte cell cycleactivation, increase mitotic index of cardiomyocytes, increasemyofilament density, increase borderzone wall thickness, or acombination thereof, wherein said cells express one or more markersidentified in Table 2 or in FIG. 5C.
 2. The composition of claim 1,wherein said cells are derived from placenta.
 3. The composition ofclaim 1, wherein said cells are progenitor cells or stem cells.
 4. Thecomposition of claim 1, wherein said cells express Cdx2, Cd9, Eomes,CD34, CD31, c-kit or a combination thereof.
 5. A composition comprisinga population of cells and a pharmaceutically acceptable carrier fortreating myocardial infarction, chronic coronary ischemia,arteriosclerosis, congestive heart failure, dilated cardiomyopathy,restenosis, coronary artery disease, heart failure, arrhythmia, angina,atherosclerosis, hypertension, or myocardial hypertrophy, wherein saidcells express one or more markers identified in Table 2 or in FIG. 5C.6. The composition of claim 5, wherein said cells are derived fromplacenta.
 7. The composition of claim 5, wherein said cells areprogenitor cells or stem cells.
 8. The composition of claim 5, whereinsaid cells express Cdx2, Cd9, Eomes, CD34, CD31, c-kit or a combinationthereof. 9-27. (canceled)
 28. A method of inducing cardiomyocyteregeneration, cardiac repair, vasculogenesis or cardiomyocytedifferentiation, comprising contacting cells with injured heart tissue,wherein said cells express one or more markers identified in Table 2 orin FIG. 5C.
 29. The method of claim 28, wherein said cells are derivedfrom placenta.
 30. The method of claim 28, wherein said cells areprogenitor cells or stem cells.
 31. The method of claim 28, wherein saidcells express Cdx2, Cd9, Eomes, CD34, CD31, c-kit or a combinationthereof.
 32. The method of claim 28, wherein the composition increasescardiomyocyte formation, increase cardiomyocyte proliferation, increasecardiomyocyte cell cycle activation, increase mitotic index ofcardiomyocytes, increase myofilament density, increase borderzone wallthickness, or a combination thereof.
 33. The method of claim 31, whereinsaid Cdx2 cells are fetal stem cells
 34. The method of claim 31, whereinthe Cdx2 cells are isolated cells.
 35. The method of claim 28, whereinthe subject is diagnosed with, or at risk for, myocardial infarction,chronic coronary ischemia, arteriosclerosis, congestive heart failure,dilated cardiomyopathy, restenosis, coronary artery disease, heartfailure, arrhythmia, angina, atherosclerosis, hypertension, ormyocardial hypertrophy.
 36. The method of claim 28, wherein introducingor contacting the composition comprises implanting the composition intocardiac tissue of the subject, or wherein introducing or contacting thecomposition comprises injecting the composition into the subject. 37.(canceled)
 38. The method of claim 36, wherein the cardiac tissue isselected from the group consisting of myocardium, endocardium,epicardium, connective tissue in the heart, and nervous tissue in theheart.
 39. The method of claim 28, wherein the amount of compositioncomprises from about 1×10⁸ to about 1×10² cells.
 40. The method of claim28, wherein the amount of introduced composition comprises from about1×10⁶ to about 1×10⁵ cells.